Methods of amplifying and sequencing nucleic acids

ABSTRACT

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.10/767,779, filed Jan. 28, 2004 (now U.S. Pat. No. 7,323,305, issuedJan. 29, 2008), which claims the benefit of priority to the followingapplications: U.S. Ser. No.: 60/476,602, filed Jun. 6, 2003, entitled“Method For Preparing Single-Stranded DNA Libraries (21465-507 PRO)”,U.S. Ser. No.: 60/476,504, filed Jun. 6, 2003, entitled “Bead EmulsionNucleic Acid Amplification (21465-508 PRO)”, U.S. Ser. No.: 60/443,471,filed Jan. 29, 2003, entitled “Double Ended Sequencing (21465-509PROA)”, U.S. Ser. No.: 60/476,313, filed Jun. 6, 2003, entitled “DoubleEnded Sequencing (21465-509 PROB)”, U.S. Ser. No.: 60/476,592, filedJun. 6, 2003, entitled “Methods Of Amplifying And Sequencing NucleicAcids (21465-510 PRO A)”, U.S. Ser. No.: 60/465,071, filed Apr. 23,2003, entitled “Massively Parallel High Throughput, Low Cost Sequencing(21465-510 PRO B)”, and U.S. Ser. No.: 60/497,985, filed Aug. 25, 2003,entitled “A Massively Parallel Picotiterplate-Based Platform ForDiscrete Picoliter-Scale Polymerase Chain Reactions (21465-510 PRO C).”

This application incorporates by reference the following co-pending U.S.patent applications: U.S. patent application Ser. No. 10/767,894, filedJan. 28, 2004; U.S. patent application Ser. No. 10/767,899, filed Jan.28, 2004; U.S. patent application Ser. No. 11/045,678, filed Jan. 28,2005; and U.S. patent application Ser. No. 10/768,729, filed Jan. 28,2004.

All patent and patent applications referred to in this application arehereby fully incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a method and apparatus for determining thebase sequences of DNA. More particularly, this invention relates tomethods and an apparatus with which the base sequences of a genome canbe amplified and determined automatically or semiautomatically.

BACKGROUND OF THE INVENTION

Development of rapid and sensitive nucleic acid sequencing methodsutilizing automated DNA sequencers has revolutionized modern molecularbiology. Analysis of entire genomes of plants, fungi, animals, bacteria,and viruses is now possible with a concerted effort by a series ofmachines and a team of technicians. However, the goal of rapid andautomated or semiautomatic sequencing of a genome in a short time hasnot been possible. There continues to be technical problems for accuratesample preparation, amplification and sequencing.

One technical problem which hinders sequence analysis of genomes hasbeen the inability of the investigator to rapidly prepare numerousnucleic acid sample encompassing a complete genome in a short period oftime.

Another technical problem is the inability to representatively amplifieda genome to a level that is compatible with the sensitivity of currentsequencing methods. Modern economically feasible sequencing machines,while sensitive, still require in excess of one million copies of a DNAfragment for sequencing. Current methods for providing high copies ofDNA sequencing involves variations of cloning or in vitro amplificationwhich cannot amplify the number of individual clones (600,000 or more,and tens of millions for a human genome) necessary for sequencing awhole genome economically.

Yet another technical problem in the limitation of current sequencingmethods which can perform, at most, one sequencing reaction perhybridization of oligonucleotide primer. The hybridization of sequencingprimers is often the rate limiting step constricting the throughput ofDNA sequencers.

In most cases, Polymerase Chain Reaction (PCR; Saiki, R. K., et al.,Science 1985, 230, 1350-1354; Mullis, K., et al., Cold Spring Harb.Symp. Quant. Biol. 1986, 51 Pt 1, 263-273) plays an integral part inobtaining DNA sequence information, amplifying minute amounts ofspecific DNA to obtain concentrations sufficient for sequencing. Yet,scaling current PCR technology to meet the increasing demands of moderngenetics is neither cost effective nor efficient, especially when therequirements for full genome sequencing are considered.

Efforts to maximize time and cost efficiencies have typically focused ontwo areas: decreasing the reaction volume required for amplificationsand increasing the number of simultaneous amplifications performed.Miniaturization confers the advantage of lowered sample and reagentutilization, decreased amplification times and increased throughputscalability.

While conventional thermocyclers require relatively long cycling timesdue to thermal mass restrictions (Woolley, A. T., et al, Anal. Chem.1996, 68, 4081-4086), smaller reaction volumes can be cycled morerapidly. Continuous flow PCR devices have utilized etched microchannelsin conjunction with fixed temperature zones to reduce reaction volumesto sub-microliter sample levels (Lagally, E. T., et al., AnalyticalChemistry 2001, 73, 565-570; Schneegas, I., et al., Lab on a Chip—TheRoyal Society of Chemistry 2001, 1, 42-49).

Glass microcapillaries, heated by air (Kalinina, O., et al., NucleicAcids Res. 1997, 25, 1999-2004) or infrared light (Oda, R. P., et al.,Anal. Chem. 1998, 70, 4361-4368; Huhmer, A. F. and Landers, J. P., Anal.Chem. 2000, 72, 5507-5512), have also served as efficient vessels fornanoliter scale reactions. Similar reaction volumes have been attainedwith microfabricated silicon thermocyclers (Burns, M. A., et al., Proc.Natl. Acad. Sci. USA 1996, 93, 5556-5561).

In many cases, these miniaturizations have reduced total PCR reactiontimes to less than 30 minutes for modified electric heating elements(Kopp, M. U., et al., Science 1998, 280, 1046-1048; Chiou, J.,Matsudaira, P., Sonin, A. and Ehrlich, D., Anal. Chem. 2001, 73,2018-2021) and hot air cyclers (Kalinina, O., et al., Nucleic Acids Res.1997, 25, 1999-2004), and to 240 seconds for some infrared controlledreactions (Giordano, B. C., et al., Anal. Biochem. 2001, 291, 124-132).

Certain technologies employ increased throughput and miniaturizationsimultaneously; as in the 1536-well system design by Sasaki et al.(Sasaki, N., et al., DNA Res. 1997, 4, 387-391), which maintainedreaction volumes under 1 μl. As another example, Nagai et al. (Nagai,H., et al, Biosens. Bioelectron. 2001, 16, 1015-1019; Nagai, H., et al.,Anal. Chem. 2001, 73, 1043-1047) reported amplification of a single testfragment in ten thousand 86 μl reaction pits etched in a single siliconwafer. Unfortunately, recovery and utilization of the amplicon fromthese methods have proven problematic, requiring evaporation throughselectively permeable membranes.

Despite these remarkable improvements in reactions volumes and cycletimes, none of the previous strategies have provided the massivelyparallel amplification required to dramatically increase throughput tolevels required for analysis of the entire human genome. DNA sequencerscontinue to be slower and more expensive than would be desired. In thepure research setting it is perhaps acceptable if a sequencer is slowand expensive. But when it is desired to use DNA sequencers in aclinical diagnostic setting such inefficient sequencing methods areprohibitive even for a well financed institution. The large-scaleparallel sequencing of thousands of clonally amplified targets wouldgreatly facilitate large-scale, whole genome library analysis withoutthe time consuming sample preparation process and expensive, error-pronecloning processes. Successful high capacity, solid-phase, clonal DNAamplification can be used for numerous applications. Accordingly, it isclear that there exists a need for preparation of a genome or largetemplate nucleic acids for sequencing, for amplification of the nucleicacid template, and for the sequencing of the amplified template nucleicacids without the constraint of one sequencing reaction perhybridization. Furthermore, there is a need for a system to connectthese various technologies into a viable automatic or semiautomaticsequencing machine.

BRIEF SUMMARY OF THE INVENTION

This invention describes an integrated system, comprising novel methodsand novel apparatus for (1) nucleic acid sample preparation, (2) nucleicacid amplification, and (3) DNA sequencing.

The invention provides a novel method for preparing a library ofmultiple DNA sequences, particularly derived from large template DNA orwhole (or partial) genome DNA. Sequences of single stranded DNA areprepared from a sample of large template DNA or whole or partial DNAgenomes through fragmentation, polishing, adaptor ligation, nick repair,and isolation of single stranded DNA. The method provides for generatinga ssDNA library linked to solid supports comprising: (a) generating alibrary of ssDNA templates; (b) attaching the ssDNA templates to solidsupports; and (c) isolating the solid supports on which one ssDNAtemplate is attached.

The invention also provides for a method of amplifying each individualmember of a DNA library in a single reaction tube, by, e.g.,encapsulating a plurality of DNA samples individually in a microcapsuleof an emulsion, performing amplification of the plurality ofencapsulated nucleic acid samples simultaneously, and releasing saidamplified plurality of DNA from the microcapsules for subsequentreactions. In one embodiment, single copies of the nucleic acid templatespecies are hybridized to DNA capture beads, suspended in completeamplification solution and emulsified into microreactors (typically 100to 200 microns in diameter), after which amplification (e.g., PCR) isused to clonally increase copy number of the initial template species tomore than 1,000,000 copies of a single nucleic acid sequence, preferablybetween 2 and 20 million copies of a single nucleic acid. Theamplification reaction, for example, may be performed simultaneouslywith at least 3,000 microreactors per microliter of reaction mix, andmay be performed with over 300,000 microreactors in a single 100 μlvolume test tube (e.g., a PCR reaction tube). The present invention alsoprovides for a method of enriching for those beads that contains asuccessful DNA amplification event (i.e., by removing beads that have noDNA attached thereto).

The invention also provides for a method of sequencing a nucleic acidfrom multiple primers with a single primer hybridization step. Two ormore sequencing primers are hybridized to the template DNA to besequenced. All the sequencing primers are then protected except for one.Sequencing (e.g., pyrophosphate sequencing) is performed again byelongating the unprotected primer. The elongation is either allowed togo to completion (with additional polymerase and dNTPs if necessary) oris terminated (by polymerase and ddNTPs). Chain completion and/ortermination reagents are removed. Then one of the protected primers isunprotected and sequencing is performed by elongating the newlyunprotected primer. This process is continued until all the sequencingprimers are deprotected and sequenced. In a preferred embodiment, twoprimers (one protected and one unprotected) are used to sequence bothends of a double stranded nucleic acid.

The invention also provides an apparatus and methods for sequencingnucleic acids using a pyrophosphate based sequencing approach. Theapparatus has a charge coupled device (CCD) camera, microfluidicschamber, sample cartridge holder, pump and flow valves. The apparatususes chemiluminescence as the detection method, which for pyrophosphatesequencing has an inherently low background. In a preferred embodiment,the sample cartridge for sequencing is termed the ‘PicoTiter plate,’ andis formed from a commercial fiber optics faceplate, acid-etched to yieldhundreds of thousands of very small wells, each well volume of 75 pL.The apparatus includes a novel reagent delivery cuvette adapted for usewith the arrays described herein, to provide fluid reagents to thepicotiter plate, and a reagent delivery means in communication with thereagent delivery cuvette. Photons from each well on the picotiter plateare channeled into specific pixels on the CCD camera to detectsequencing reactions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic representation of the entire process oflibrary preparation including the steps of template DNA fragmentation(FIG. 1A), end polishing (FIG. 1B), adaptor ligation (FIG. 1C), nickrepair, strand extension and gel isolation (FIG. 1D). FIG. 1E depicts aschematic representation of the stages for amplification and sequencingof template DNA (FIG. 1E). FIG. 1F depicts a representative agarose gelcontaining a sample preparation of a 180-350 base pair adenovirus DNAlibrary according to the methods of this invention. FIG. 1G depicts adetailed schematic representation of library preparation, amplification,and sequencing.

FIG. 2A depicts a schematic representation of the universal adaptordesign according to the present invention. Each universal adaptor isgenerated from two complementary ssDNA oligonucleotides that aredesigned to contain a 20 bp nucleotide sequence for PCR priming, a 20 bpnucleotide sequence for sequence priming and a unique 4 bpdiscriminating sequence comprised of a non-repeating nucleotide sequence(i.e., ACGT, CAGT, etc.). FIG. 2B depicts a representative universaladaptor sequence pair for use with the invention. Adaptor A sensestrand: SEQ ID NO:1; Adaptor A antisense strand: SEQ ID NO:2; Adaptor Bsense strand: SEQ ID NO:3; Adaptor B antisense strand: SEQ ID NO:4. FIG.2C depicts a schematic representation of universal adaptor design foruse with the invention.

FIG. 3 depicts the strand displacement and extension of nickeddouble-stranded DNA fragments according to the present invention.Following the ligation of universal adaptors generated from syntheticoligonucleotides, double-stranded DNA fragments will be generated thatcontain two nicked regions following T4 DNA ligase treatment (FIG. 3A).The addition of a strand displacing enzyme (i.e., Bst DNA polymerase I)will bind nicks (FIG. 3B), strand displace the nicked strand andcomplete nucleotide extension of the strand (FIG. 3C) to producenon-nicked double-stranded DNA fragments (FIG. 3D).

FIG. 4 depicts the isolation of directionally-ligated single-strandedDNA according to the present invention using streptavidin-coated beads.Following ligation with universal adaptors A and B (the two differentadaptors are sometimes referred to as a “first” and “second” universaladaptor), double-stranded DNA will contain adaptors in four possiblecombinations: AA, BB, AB and BA. When universal adaptor B contains a5′-biotin, magnetic streptavidin-coated solid supports are used tocapture and isolate the AB, BA and BB populations (population AA iswashed away). The BB population is retained on the beads as each end ofthe double-stranded DNA is attached to a bead and is not released.However, upon washing in the presence of a low salt buffer, onlypopulations AB and BA will release a single-stranded DNA fragment thatis complementary to the bound strand. Single-stranded DNA fragments areisolated from the supernatant and used as template for subsequentamplification and sequencing. This method is described below in moredetail.

FIG. 5 depicts a schematic of the structure of a DNA capture bead.

FIG. 6A-B depicts a schematic of one embodiment of a bead emulsionamplification process.

FIG. 7 depicts a schematic of an enrichment process to remove beads thatdo not have any DNA attached thereto.

FIG. 8A-J depict a schematic representation of the double endedsequencing reaction according to the present invention.

FIG. 9 depicts a double-ended sequencing demonstration on apyrosequencing apparatus according to the invention.

FIGS. 10A-F depict an exemplary double ended sequencing process. FIG.10F shows SEQ ID NO:44: atgcacatggttgacacagtggt; SEQ ID NO:45:atgcacatggttgacacagtgg; SEQ ID NO:46: atgccaccgacctagtctcaaactt.

FIGS. 11A-D depict schematic illustrations of rolling circle-basedamplification using an anchor primer. FIG. 11B shows a template moleculehaving the sequence 5′-tcg tgt gag gtc tca gca tct tat gta tat tta cttcta ttc tca gtt gcc taa gct gca gcc a-3′(SEQ ID NO:5) annealed to ananchor primer having a biotin linker at its 5′ terminus and the sequence5′-gac ctc aca cga tgg ctg cag ctt-3′(SEQ ID NO:6). FIG. 11C shows ageneric anchor primer having the sequence 5′-gac ctc aca cga tgg ctg cagctt-3′(SEQ ID NO:7). The anchor primer anneals to an SNP probe havingthe sequence 5′-ttt ata tgt att cta cga ctc tgg agt gtg cta ccg acg tcgaat ccg ttg act ctt atc ttc a-3′(SEQ ID NO:8). The SNP probe in turnhybridizes to a region of a SNP-containing region of a gene having thesequence 5′-cta gct cgt aca tat aaa tga aga taa gat cct g-3′(SEQ IDNO:9). FIG. 11D shows an anchor primer having the sequence 5′-gac ctcaca cga gta gca tgg ctg cag ctt-3′(SEQ ID NO:10) is attached to asurface through a biotin linker. A template molecule having the sequence5′-tcg tgt gag gtc tca gca tct tat gta tat tta ctt cta ttc tca gtt gcctaa gct gca gcc a-3′(SEQ ID NO:11) is annealed to the anchor primer toresult in partially single stranded, or gapped region, in the anchorprimer flanked by a double-stranded region.

FIG. 12 depicts a drawing of a sequencing apparatus according to thepresent invention.

FIG. 13 depicts a drawing of a reagent delivery/perfusion chamberaccording to the present invention.

FIG. 14 depicts a micrograph of a cavitated fiber optic bundle, termed aPicoTiter Plate™, of the invention.

FIG. 15 depicts a micrograph of a picotiter plate carpeted with beadshaving DNA template immobilized thereon and sulfurylase and luciferaseimmobilized thereon.

FIG. 16 depicts a schematic illustration of the reagent flow chamber andFORA (PicoTiter Plate™).

FIG. 17 depicts a diagram of the analytical instrument of the presentinvention.

FIG. 18 depicts a schematic illustration of microscopic parallelsequencing reactions within a PicoTiter Plate™.

FIG. 19 depicts a micrograph of single well reactions.

FIG. 20 depicts a PicoTiterPlate™ loading cartridge. “A” refers to aPicoTiterPlate™ with microwells facing into the cartridge, the distancebetween the open sides of the PicoTiterPlate™ wells and the wall of theloading cartridge is 0.3 mm; “B” refers to a silicon sealing gasket; “C”refers to an inlet port; “D” refers to an inlet loading tube; “E” refersto an outlet port and “F” refers to an outlet tube. The PicoTiterPlate™is held in the cartridge with plastic clamps. The liquid is filled viathe inlet loading tube D and enters the space between the open sides ofthe PicoTiterPlate™ wells and the wall of the loading cartridge throughthe inlet port C. The area defined by the silicon sealing gasket B isfilled and excess liquid leaves the cartridge via the outlet port E andthe outlet tubing F.

FIG. 21 depicts a PicoTiterPlate™ amplification chamber in explodedview. “A” refers to an amplification chamber lid with six retainingbolts; “B” refers to a closed cell foam insulation pad; “C” refers to a25 mm by 75 mm standard glass microscope slide; “D” refers to a 0.25 mmthick silicon sheet; “E” refers to a PicoTiterPlate™; “F” refers to anamplification chamber base; “G” refers to a second 0.25 mm thick siliconsheet.

FIG. 22 depicts a schematic diagram of solid phase PicoTiterPlate™ PCR.The cylindrical structures symbolize individual PicoTiterPlate™ wells.Gray spheres symbolize beads with immobilized primers. Forward “F” (red)and Reverse “R” (blue) primers are shown in 5′ to 3′ orientation asindicated by arrows. Synthesized sequence complementary to the Forwardand Reverse primers are shown as dark red (F complement) and dark blue(R complement) bars. Single stranded template DNA is shown as solid grayline, newly synthesized DNA strands as dashed gray lines. Fluorescentlylabeled hybridization probes are shown as green bars.

FIGS. 23A-C depict fluorescent probes hybridization to bead-immobilizedtest DNA fragments. FIG. 23A (upper left) and 23B (upper right)illustrate the specificity of a mixed population of probes hybridized tofragment A and fragment B immobilized on control beads, respectively.Fragment B beads exhibited the Alexa Fluor 647 signal (red), and thefragment A beads exhibited the Alexa Fluor 488 signal (green). FIG. 23C(bottom panel) depicts probe fluorescence from DNA capture beads afterPTPCR. Beads displayed homogenous fragment A and fragment B signals, aswell as mixes of templates, shown as varying degrees of yellow.

FIG. 24 depicts representative BioAnalyzer output from analysis of asingle stranded DNA library.

FIG. 25 depicts an insert flanked by PCR primers and sequencing primers.

FIG. 26 depicts truncated product produced by PCR primer mismatch atcross-hybridization region (CHR).

FIG. 27 depicts the calculation for primer candidates based on meltingtemperature.

FIGS. 28A-D depict the assembly for the nebulizer used for the methodsof the invention. A tube cap was placed over the top of the nebulizer(FIG. 7A) and the cap was secured with a nebulizer clamp assembly (FIG.7B). The bottom of the nebulizer was attached to the nitrogen supply(FIG. 7C) and the entire device was wrapped in parafilm (FIG. 7D).

FIG. 29A depicts representative results for LabChip analysis of a singlestranded DNA library following nebulization and polishing.

FIG. 29B depicts representative size distribution results for anadaptor-ligated single stranded DNA library following nebulization,polishing, and gel purification.

FIG. 30 depiction of jig used to hold tubes on the stir plate belowvertical syringe pump. The jig was modified to hold three sets of beademulsion amplification reaction mixtures. The syringe was loaded withthe PCR reaction mixture and beads.

FIG. 31 depiction of optimal placement of syringes in vertical syringepump and orientation of emulsion tubes below syringe outlets.

FIG. 32 depiction of optimal placement of syringe pump pusher blockagainst syringe plungers, and optimal orientation of jig on the stirplate. Using this arrangement, the syringe contents were expelled intothe agitated emulsion oil.

FIG. 33 depiction of beads (see arrows) suspended in individualmicroreactors according to the methods of the invention.

FIG. 34 depiction of double ended sequencing results showing that thesequence of both ends of a DNA template are determined. SEQ ID NO:44:atgcacatggttgacacagtggt; SEQ ID NO:45: atgcacatggttgacacagtgg; SEQ IDNO:46: atgccaccgacctagtctcaaactt.

FIG. 35A-C illustrates the encapsulation of a bead comprising twooligonucleotide sequences for double stranded sequencing.

FIG. 36 illustrates solution phase PCR and drive to bead procedure—astep in a preferred embodiment of double ended sequencing.

FIG. 37A-E illustrates emulsion breaking and recovery of amplifiedtemplate DNA on a bead—a step in a preferred embodiment of double endedsequencing.

FIG. 38 depicts a schematic representation of a preferred method ofdouble stranded sequencing.

FIG. 39A-B illustrates the results of sequencing a Staphylococcus aureusgenome.

FIG. 40 illustrates the average read lengths in one experiment involvingdouble ended sequencing.

FIG. 41 illustrates the number of wells for each genome span in a doubleended sequencing experiment.

FIG. 42 illustrates a typical output and alignment string from a doubleended sequencing procedure. Sequences shown in order, from top tobottom: SEQ ID NO:47-SEQ ID NO:60.

DETAILED DESCRIPTION OF THE INVENTION

A novel platform is described herein which permits simultaneousamplification of three hundred thousand discrete PCR reactions (PTPCR)in volumes as low as 39.5 picoliters. The pooled PTPCR products from theentire reaction can be recovered through a wash step and assayed viareal-time PCR for the presence and abundance of specific templates. Ofgreater interest, it is shown herein that these PTPCR products can bedriven to solid supports and detected by hybridization with two colorfluorescent probes, allowing high capacity, solid-phase, clonal DNAamplification and large-scale parallel sequencing.

The present invention is directed to a method and apparatus forperforming genomic sequencing which satisfies the objectives of (1)preparing a nucleic acid (e.g., a genome) in a rapid and efficientmanner for sequencing, (2) amplifying the nucleic acid in arepresentative manner, and (3) performing multiple sequencing reactionswith only one primer hybridization. The present invention isparticularly suited for genotyping, detection and diagnosis from a smallsample of nucleic acid in a cost efficient manner. Each of theseobjectives are listed below.

Definitions:

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials similaror equivalent to those described herein can be used in the practice ofthe present invention, and exemplified suitable methods and materialsare described below. For example, methods may be described whichcomprise more than two steps. In such methods, not all steps may berequired to achieve a defined goal and the invention envisions the useof isolated steps to achieve these discrete goals. The disclosures ofall publications, patent applications, patents and other references areincorporated in toto herein by reference. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

As used herein, the term “universal adaptor” refers to two complementaryand annealed oligonucleotides that are designed to contain a nucleotidesequence for PCR priming and a nucleotide sequence for sequence priming.Optionally, the universal adaptor may further include a uniquediscriminating key sequence comprised of a non-repeating nucleotidesequence (i.e., ACGT, CAGT, etc.). A set of universal adaptors comprisestwo unique and distinct double-stranded sequences that can be ligated tothe ends of double-stranded DNA. Therefore, the same universal adaptoror different universal adaptors can be ligated to either end of the DNAmolecule. When comprised in a larger DNA molecule that is singlestranded or when present as an oligonucleotide, the universal adaptormay be referred to as a single stranded universal adaptor.

“Target DNA” shall mean a DNA whose sequence is to be determined by themethods and apparatus of the invention.

Binding pair shall mean a pair of molecules that interact by means ofspecific non-covalent interactions that depend on the three-dimensionalstructures of the molecules involved. Typical pairs of specific bindingpartners include antigen-antibody, hapten-antibody, hormone-receptor,nucleic acid strand-complementary nucleic acid strand, substrate-enzyme,substrate analog-enzyme, inhibitor-enzyme, carbohydrate-lectin,biotin-avidin, and virus-cellular receptor.

As used herein, the term “discriminating key sequence” refers to asequence consisting of at least one of each of the fourdeoxyribonucleotides (i.e., A, C, G, T). The same discriminatingsequence can be used for an entire library of DNA fragments.Alternatively, different discriminating key sequences can be used totrack libraries of DNA fragments derived from different organisms.

As used herein, the term “plurality of molecules” refers to DNA isolatedfrom the same source, whereby different organisms may be preparedseparately by the same method. In one embodiment, the plurality of DNAsamples is derived from large segments of DNA, whole genome DNA, cDNA,viral DNA or from reverse transcripts of viral RNA. This DNA may bederived from any source, including mammal (i.e., human, nonhumanprimate, rodent or canine), plant, bird, reptile, fish, fungus, bacteriaor virus.

As used herein, the term “library” refers to a subset of smaller sizedDNA species generated from a single DNA template, either segmented orwhole genome.

As used herein, the term “unique”, as in “unique PCR priming regions”refers to a sequence that does not exist or exists at an extremely lowcopy level within the DNA molecules to be amplified or sequenced.

As used herein, the term “compatible” refers to an end of doublestranded DNA to which an adaptor molecule may be attached (i.e., bluntend or cohesive end).

As used herein, the term “fragmenting” refers to a process by which alarger molecule of DNA is converted into smaller pieces of DNA.

As used herein, “large template DNA” would be DNA of more than 25 kb,preferably more than 500 kb, more preferably more than 1 MB, and mostpreferably 5 MB or larger.

As used herein, the term “stringent hybridization conditions” refers tothose conditions under which only complimentary sequences will hybridizeto each other.

The invention described here is generally a system and methods forprocessing nucleic acids. The system and methods can be used to processnucleic acids in a multitude of ways that utilize sequencing of nucleicacids. Such sequencing can be performed to determine the identity of asequence of nucleic acids, or for single nucleotide polymorphismdetection in nucleic acid fragments, for nucleic acid expressionprofiling (comparing the nucleic acid expression profile between two ormore states—e.g., comparing between diseased and normal tissue orcomparing between untreated tissue and tissue treated with drug,enzymes, radiation or chemical treatment), for haplotying (comparinggenes or variations in genes on each of the two alleles present in ahuman subject), for karyotyping (diagnostically comparing one or moregenes in a test tissue—typically from an embryo/fetus prior toconception to detect birth defects—with the same genes from “normal”karyotyped subjects), and for genotyping (comparing one or more genes ina first individual of a species with the same genes in other individualsof the same species).

The system has a number of components. These include (1) the nucleicacid template that is to be processed, (2) a picotiter plate forcontaining the nucleic acid template, (3) a flow chamber and fluiddelivery means that permits flow of nucleic acid processing reagentsover the nucleic acid template where the processing reagents generatelight as the nucleic acid is processed, (4) a light capture means thatdetects light emitted as the nucleic acid is processed and that convertsthe captured light into data, and (5) data processing means thatprocesses the data to yield meaningful information about the nucleicacid that has been processed. Each of these components of the systemwill be discussed in detail below.

1. Nucleic Acid Template and Preparation Thereof

Nucleic Acid Templates

The nucleic acid templates that can be sequenced according to theinvention, e.g., a nucleic acid library, in general can include opencircular or closed circular nucleic acid molecules. A “closed circle” isa covalently closed circular nucleic acid molecule, e.g., a circular DNAor RNA molecule. An “open circle” is a linear single-stranded nucleicacid molecule having a 5′ phosphate group and a 3′ hydroxyl group.

In one embodiment, the single stranded nucleic acid contains at least100 copies of a specific nucleic acid sequence, each copy covalentlylinked end to end. In some embodiments, the open circle is formed insitu from a linear double-stranded nucleic acid molecule. The ends of agiven open circle nucleic acid molecule can be ligated by DNA ligase.Sequences at the 5′ and 3′ ends of the open circle molecule arecomplementary to two regions of adjacent nucleotides in a second nucleicacid molecule, e.g., an adapter region of an anchor primer (sometimesreferred to as an adapter), or to two regions that are nearly adjoiningin a second DNA molecule. Thus, the ends of the open-circle molecule canbe ligated using DNA ligase, or extended by DNA polymerase in agap-filling reaction. Open circles are described in detail in Lizardi,U.S. Pat. No. 5,854,033, fully incorporated herein by reference. An opencircle can be converted to a closed circle in the presence of a DNAligase (for DNA) or RNA ligase following, e.g., annealing of the opencircle to an anchor primer.

If desired, nucleic acid templates can be provided as padlock probes.Padlock probes are linear oligonucleotides that includetarget-complementary sequences located at each end, and which areseparated by a linker sequence. The linkers can be ligated to ends ofmembers of a library of nucleic acid sequences that have been, e.g.,physically sheared or digested with restriction endonucleases. Uponhybridization to a target-sequence, the 5′- and 3′-terminal regions ofthese linear oligonucleotides are brought in juxtaposition. Thisjuxtaposition allows the two probe segments (if properly hybridized) tobe covalently-bound by enzymatic ligation (e.g., with T4 DNA ligase),thus converting the probes to circularly-closed molecules which arecatenated to the specific target sequences (see e.g., Nilsson, et al.,1994. Science 265: 2085-2088). The resulting probes are suitable for thesimultaneous analysis of many gene sequences both due to theirspecificity and selectivity for gene sequence variants (see e.g.,Lizardi, et al., 1998. Nat. Genet. 19: 225-232; Nilsson, et al., 1997.Nat. Genet. 16: 252-255) and due to the fact that the resulting reactionproducts remain localized to the specific target sequences. Moreover,intramolecular ligation of many different probes is expected to be lesssusceptible to non-specific cross-reactivity than multiplex PCR-basedmethodologies where non-cognate pairs of primers can give rise toirrelevant amplification products (see e.g., Landegren and Nilsson,1997. Ann. Med. 29: 585-590).

A starting nucleic acid template library can be constructed comprisingeither single-stranded or double-stranded nucleic acid molecules,provided that the nucleic acid sequence includes a region that, ifpresent in the library, is available for annealing, or can be madeavailable for annealing, to an anchor primer sequence. For example, whenused as a template for rolling circle amplification, a region of adouble-stranded template needs to be at least transientlysingle-stranded in order to act as a template for extension of theanchor primer.

Library templates can include multiple elements, including, but notlimited to, one or more regions that are complementary to the anchorprimer. For example, the template libraries may include a regioncomplementary to a sequencing primer, a control nucleotide region, andan insert sequence comprised of the sequencing template to besubsequently characterized. As is explained in more detail below, thecontrol nucleotide region is used to calibrate the relationship betweenthe amount of byproduct and the number of nucleotides incorporated. Asutilized herein the term “complement” refers to nucleotide sequencesthat are able to hybridize to a specific nucleotide sequence to form amatched duplex.

In one embodiment, a library template includes: (i) two distinct regionsthat are complementary to the anchor primer, (ii) one region homologousto the sequencing primer, (iii) one optional control nucleotide region,(iv) an insert sequence of, e.g., 30-500, 50-200, or 60-100 nucleotides,that is to be sequenced. The template can, of course, include two,three, or all four of these features.

The template nucleic acid can be constructed from any source of nucleicacid, e.g., any cell, tissue, or organism, and can be generated by anyart-recognized method. Suitable methods include, e.g., sonication ofgenomic DNA and digestion with one or more restriction endonucleases(RE) to generate fragments of a desired range of lengths from an initialpopulation of nucleic acid molecules. Preferably, one or more of therestriction enzymes have distinct four-base recognition sequences.Examples of such enzymes include, e.g., Sau3A1, MspI, and TaqI.Preferably, the enzymes are used in conjunction with anchor primershaving regions containing recognition sequences for the correspondingrestriction enzymes. In some embodiments, one or both of the adapterregions of the anchor primers contain additional sequences adjoiningknown restriction enzyme recognition sequences, thereby allowing forcapture or annealing to the anchor primer of specific restrictionfragments of interest to the anchor primer. In other embodiments, therestriction enzyme is used with a type IIS restriction enzyme.

Alternatively, template libraries can be made by generating acomplementary DNA (cDNA) library from RNA, e.g., messenger RNA (mRNA).The cDNA library can, if desired, be further processed with restrictionendonucleases to obtain a 3′ end characteristic of a specific RNA,internal fragments, or fragments including the 3′ end of the isolatedRNA. Adapter regions in the anchor primer may be complementary to asequence of interest that is thought to occur in the template library,e.g., a known or suspected sequence polymorphism within a fragmentgenerated by endonuclease digestion.

In one embodiment, an indexing oligonucleotide can be attached tomembers of a template library to allow for subsequent correlation of atemplate nucleic acid with a population of nucleic acids from which thetemplate nucleic acid is derived. For example, one or more samples of astarting DNA population can be fragmented separately using any of thepreviously disclosed methods (e.g., restriction digestion, sonication).An indexing oligonucleotide sequence specific for each sample isattached to, e.g., ligated to, the termini of members of the fragmentedpopulation. The indexing oligonucleotide can act as a region forcircularization, amplification and, optionally, sequencing, whichpermits it to be used to index, or code, a nucleic acid so as toidentify the starting sample from which it is derived.

Distinct template libraries made with a plurality of distinguishableindexing primers can be mixed together for subsequent reactions.Determining the sequence of the member of the library allows for theidentification of a sequence corresponding to the indexingoligonucleotide. Based on this information, the origin of any givenfragment can be inferred.

The invention includes a sample preparation process that results in asolid or a mobile solid substrate array containing a plurality of anchorprimers or adapters covalently linked to template nucleic acids.

When the template nucleic acid is circular, formation of the covalentlylinked anchor primer and one or more copies of the target nucleic acidpreferably occurs by annealing the anchor primer to a complementaryregion of a circular nucleic acid, and then extending the annealedanchor primer with a polymerase to result in formation of a nucleic acidcontaining one or more copies of a sequence complementary to thecircular nucleic acid.

Attachment of the anchor primer to a solid or mobile solid substrate canoccur before, during, or subsequent to extension of the annealed anchorprimer. Thus, in one embodiment, one or more anchor primers are linkedto the solid or a mobile solid substrate, after which the anchor primeris annealed to a target nucleic acid and extended in the presence of apolymerase. Alternatively, in a second embodiment, an anchor primer isfirst annealed to a target nucleic acid, and a 3′OH terminus of theannealed anchor primer is extended with a polymerase. The extendedanchor primer is then linked to the solid or mobile solid substrate. Byvarying the sequence of anchor primers, it is possible to specificallyamplify distinct target nucleic acids present in a population of nucleicacids.

Outlined below is a preferred embodiment for the preparation of templatenucleic acids for amplification and sequencing reactions. The inventionincludes a method for preparing the sample DNA comprised of sevengeneral steps: (a) fragmenting large template DNA or whole genomic DNAsamples to generate a plurality of digested DNA fragments; (b) creatingcompatible ends on the plurality of digested DNA samples; (c) ligating aset of universal adaptor sequences onto the ends of fragmented DNAmolecules to make a plurality of adaptor-ligated DNA molecules, whereineach universal adaptor sequence has a known and unique base sequencecomprising a common PCR primer sequence, a common sequencing primersequence and a discriminating four base key sequence and wherein oneadaptor is attached to biotin; (d) separating and isolating theplurality of ligated DNA fragments; (e) removing any portion of theplurality of ligated DNA fragments; (f) nick repair and strand extensionof the plurality of ligated DNA fragments; (g) attaching each of theligated DNA fragments to a solid support; and (h) isolating populationscomprising single-stranded adaptor-ligated DNA fragments for which thereis a unique adaptor at each end (i.e., providing directionality).

The following discussion summarizes the basic steps involved in themethods of the invention. The steps are recited in a specific order,however, as would be known by one of skill in the art, the order of thesteps may be manipulated to achieve the same result. Such manipulationsare contemplated by the inventors. Further, some steps may be minimizedas would also be known by one of skill in the art.

Fragmentation

In the practice of the methods of the present invention, thefragmentation of the DNA sample can be done by any means known to thoseof ordinary skill in the art. Preferably, the fragmenting is performedby enzymatic or mechanical means. The mechanical means may be sonicationor physical shearing. The enzymatic means may be performed by digestionwith nucleases (e.g., Deoxyribonuclease I (DNase I)) or one or morerestriction endonucleases. In a preferred embodiment, the fragmentationresults in ends for which the sequence is not known.

In a preferred embodiment, the enzymatic means is DNase I. DNase I is aversatile enzyme that nonspecifically cleaves double-stranded DNA(dsDNA) to release 5′-phosphorylated di-, tri-, and oligonucleotideproducts. DNase I has optimal activity in buffers containing Mn²⁺,Mg^(n+) and Ca²⁺, but no other salts. The purpose of the DNase Idigestion step is to fragment a large DNA genome into smaller speciescomprising a library. The cleavage characteristics of DNase I willresult in random digestion of template DNA (i.e., no sequence bias) andin the predominance of blunt-ended dsDNA fragments when used in thepresence of manganese-based buffers (Melgar, E. and D. A. Goldthwait.1968. Deoxyribonucleic acid nucleases. II. The effects of metal on themechanism of action of deoxyribonuclease I. J. Biol. Chem. 243: 4409).The range of digestion products generated following DNase I treatment ofgenomic templates is dependent on three factors: i) amount of enzymeused (units); ii) temperature of digestion (° C.); and iii) incubationtime (minutes). The DNase I digestion conditions outlined below havebeen optimized to yield genomic libraries with a size range from 50-700base pairs (bp).

In a preferred embodiment, the DNase I digests large template DNA orwhole genome DNA for 1-2 minutes to generate a population ofpolynucleotides. In another preferred embodiment, the DNase I digestionis performed at a temperature between 10° C.-37° C. In yet anotherpreferred embodiment, the digested DNA fragments are between 50 bp to700 bp in length.

Polishing

Digestion of genomic DNA (gDNA) templates with DNase I in the presenceof Mn²⁺ will yield fragments of DNA that are either blunt-ended or haveprotruding termini with one or two nucleotides in length. In a preferredembodiment, an increased number of blunt ends are created with Pfu DNApolymerase. In other embodiments, blunt ends can be created with lessefficient DNA polymerases such as T4 DNA polymerase or Klenow DNApolymerase. Pfu “polishing” or blunt ending is used to increase theamount of blunt-ended species generated following genomic templatedigestion with DNase I. Use of Pfu DNA polymerase for fragment polishingwill result in the fill-in of 5′ overhangs. Additionally, Pfu DNApolymerase does not exhibit DNA extendase activity but does have 3′→5′exonuclease activity that will result in the removal of single anddouble nucleotide extensions to further increase the amount ofblunt-ended DNA fragments available for adaptor ligation (Costa, G. L.and M. P. Weiner. 1994a. Protocols for cloning and analysis ofblunt-ended PCR-generated DNA fragments. PCR Methods Appl 3(5):S95;Costa, G. L., A. Grafsky and M. P. Weiner. 1994b. Cloning and analysisof PCR-generated DNA fragments. PCR Methods Appl 3(6):338; Costa, G. L.and M. P. Weiner. 1994c. Polishing with T4 or Pfu polymerase increasesthe efficiency of cloning of PCR products. Nucleic Acids Res.22(12):2423).

Adaptor Ligation

If the libraries of nucleic acids are to be attached to the solidsubstrate, then preferably the nucleic acid templates are annealed toanchor primer sequences using recognized techniques (see, e.g., Hatch,et al., 1999. Genet. Anal. Biomol. Engineer. 15: 35-40; Kool, U.S. Pat.No. 5,714,320 and Lizardi, U.S. Pat. No. 5,854,033). In general, anyprocedure for annealing the anchor primers to the template nucleic acidsequences is suitable as long as it results in formation of specific,i.e., perfect or nearly perfect, complementarity between the adapterregion or regions in the anchor primer sequence and a sequence presentin the template library.

In a preferred embodiment, following fragmentation and blunt ending ofthe DNA library, universal adaptor sequences are added to each DNAfragment. The universal adaptors are designed to include a set of uniquePCR priming regions that are typically 20 bp in length located adjacentto a set of unique sequencing priming regions that are typically 20 bpin length optionally followed by a unique discriminating key sequenceconsisting of at least one of each of the four deoxyribonucleotides(i.e., A, C, G, T). In a preferred embodiment, the discriminating keysequence is 4 bases in length. In another embodiment, the discriminatingkey sequence may be combinations of 1-4 bases. In yet anotherembodiment, each unique universal adaptor is forty-four bp (44 bp) inlength. In a preferred embodiment the universal adaptors are ligated,using T4 DNA ligase, onto each end of the DNA fragment to generate atotal nucleotide addition of 88 bp to each DNA fragment. Differentuniversal adaptors are designed specifically for each DNA librarypreparation and will therefore provide a unique identifier for eachorganism. The size and sequence of the universal adaptors may bemodified as would be apparent to one of skill in the art.

For example, to prepare two distinct universal adaptors (i.e., “first”and “second”), single-stranded oligonucleotides may be ordered from acommercial vendor (i.e., Integrated DNA Technologies, Iowa or OperonTechnologies, Calif.). In one embodiment, the universal adaptoroligonucleotide sequences are modified during synthesis with two orthree phosphorothioate linkages in place of phosphodiester linkages atboth the 5′ and 3′ ends. Unmodified oligonucleotides are subject torapid degradation by nucleases and are therefore of limited utility.Nucleases are enzymes that catalyze the hydrolytic cleavage of apolynucleotide chain by cleaving the phosphodiester linkage betweennucleotide bases. Thus, one simple and widely used nuclease-resistantchemistry available for use in oligonucleotide applications is thephosphorothioate modification. In phosphorothioates, a sulfur atomreplaces a non-bridging oxygen in the oligonucleotide backbone making itresistant to all forms of nuclease digestion (i.e. resistant to bothendonuclease and exonuclease digestion). Each oligonucleotide isHPLC-purified to ensure there are no contaminating or spuriousoligonucleotide sequences in the synthetic oligonucleotide preparation.The universal adaptors are designed to allow directional ligation to theblunt-ended, fragmented DNA. Each set of double-stranded universaladaptors are designed with a PCR priming region that containsnoncomplementary 5′ four-base overhangs that cannot ligate to theblunt-ended DNA fragment as well as prevent ligation with each other atthese ends. Accordingly, binding can only occur between the 3′ end ofthe adaptor and the 5′ end of the DNA fragment or between the 3′ end ofthe DNA fragment and the 5′ end of the adaptor. Double-strandeduniversal adaptor sequences are generated by using single-strandedoligonucleotides that are designed with sequences that allow primarilycomplimentary oligonucleotides to anneal, and to preventcross-hybridization between two non-complimentary oligonucleotides. Inone embodiment, 95% of the universal adaptors are formed from theannealing of complimentary oligonucleotides. In a preferred embodiment,97% of the universal adaptors are formed from the annealing ofcomplimentary oligonucleotides. In a more preferred embodiment, 99% ofthe universal adaptors are formed from the annealing of complimentaryoligonucleotides. In a most preferred embodiment, 100% of the universaladaptors are formed from the annealing of complimentaryoligonucleotides.

One of the two adaptors can be linked to a support binding moiety. In apreferred embodiment, a 5′ biotin is added to the first universaladaptor to allow subsequent isolation of ssDNA template and noncovalentcoupling of the universal adaptor to the surface of a solid support thatis saturated with a biotin-binding protein (i.e. streptavidin,neutravidin or avidin). Other linkages are well known in the art and maybe used in place of biotin-streptavidin (for exampleantibody/antigen-epitope, receptor/ligand and oligonucleotide pairing orcomplimentarily) one embodiment, the solid support is a bead, preferablya polystyrene bead. In one preferred embodiment, the bead has a diameterof about 2.8 μm. As used herein, this bead is referred to as a “sampleprep bead”.

Each universal adaptor may be prepared by combining and annealing twossDNA oligonucleotides, one containing the sense sequence and the secondcontaining the antisense (complementary) sequence. Schematicrepresentation of the universal adaptor design is outlined in FIG. 2.

Isolation of Ligation Products

The universal adaptor ligation results in the formation of fragmentedDNAs with adaptors on each end, unbound single adaptors, and adaptordimers. In a preferred embodiment, agarose gel electrophoresis is usedas a method to separate and isolate the adapted DNA library populationfrom the unligated single adaptors and adaptor dimer populations. Inother embodiments, the fragments may be separated by size exclusionchromatography or sucrose sedimentation. The procedure of DNase Idigestion of DNA typically yields a library population that ranges from50-700 bp. In a preferred embodiment, upon conducting agarose gelelectrophoresis in the presence of a DNA marker, the addition of the 88bp universal adaptor set will shift the DNA library population to alarger size and will result in a migration profile in the size range ofapproximately 130-800 bp; adaptor dimers will migrate at 88 bp; andadaptors not ligated will migrate at 44 bp. Therefore, numerousdouble-stranded DNA libraries in sizes ranging from 200-800 bp can bephysically isolated from the agarose gel and purified using standard gelextraction techniques. In one embodiment, gel isolation of the adaptedligated DNA library will result in the recovery of a library populationranging in size from 200-400 bp. Other methods of distinguishingadaptor-ligated fragments are known to one of skill in the art.

Nick Repair

Because the DNA oligonucleotides used for the universal adaptors are not5′ phosphorylated, gaps will be present at the 3′ junctions of thefragmented DNAs following ligase treatment (see FIG. 3A). These two“gaps” or “nicks” can be filled in by using a DNA polymerase enzyme thatcan bind to, strand displace and extend the nicked DNA fragments. DNApolymerases that lack 3′→5′ exonuclease activity but exhibit 5′→3′exonuclease activity have the ability to recognize nicks, displace thenicked strands, and extend the strand in a manner that results in therepair of the nicks and in the formation of non-nicked double-strandedDNA (see FIGS. 3B and 3C) (Hamilton, S. C., J. W. Farchaus and M. C.Davis. 2001. DNA polymerases as engines for biotechnology. BioTechniques31:370).

Several modifying enzymes are utilized for the nick repair step,including but not limited to polymerase, ligase and kinase. DNApolymerases that can be used for this application include, for example,E. coli DNA pol I, Thermoanaerobacter thermohydrosulfuricus pol I, andbacteriophage phi 29. In a preferred embodiment, the strand displacingenzyme Bacillus stearothermophilus pol I (Bst DNA polymerase I) is usedto repair the nicked dsDNA and results in non-nicked dsDNA (see FIG.3D). In another preferred embodiment, the ligase is T4 and the kinase ispolynucleotide kinase.

Isolation of Single-Stranded DNA

Following the generation of non-nicked dsDNA, ssDNAs comprising both thefirst and second adaptor molecules are to be isolated (desiredpopulations are designated below with asterisks; “A” and “B” correspondto the first and second adaptors). Double-stranded DNA libraries willhave adaptors bound in the following configurations:

Universal Adaptor A—DNA fragment—Universal Adaptor A

Universal Adaptor B—DNA fragment—Universal Adaptor A*

Universal Adaptor A—DNA fragment—Universal Adaptor B*

Universal Adaptor B—DNA fragment—Universal Adaptor B

Universal adaptors are designed such that only one universal adaptor hasa 5′ biotin moiety. For example, if universal adaptor B has a 5′ biotinmoiety, streptavidin-coated sample prep beads can be used to bind alldouble-stranded DNA library species with universal adaptor B. Genomiclibrary populations that contain two universal adaptor A species willnot contain a 5′ biotin moiety and will not bind tostreptavidin-containing sample prep beads and thus can be washed away.The only species that will remain attached to beads are those withuniversal adaptors A and B and those with two universal adaptor Bsequences. DNA species with two universal adaptor B sequences (i.e.,biotin moieties at each 5′ end) will be bound to streptavidin-coatedsample prep beads at each end, as each strand comprised in the doublestrand will be bound. Double-stranded DNA species with a universaladaptor A and a universal adaptor B will contain a single 5′biotinmoiety and thus will be bound to streptavidin-coated beads at only oneend. The sample prep beads are magnetic, therefore, the sample prepbeads will remain coupled to a solid support when magnetized.Accordingly, in the presence of a low-salt (“melt” or denaturing)solution, only those DNA fragments that contain a single universaladaptor A and a single universal adaptor B sequence will release thecomplementary unbound strand. This single-stranded DNA population may becollected and quantitated by, for example, pyrophosphate sequencing,real-time quantitative PCR, agarose gel electrophoresis or capillary gelelectrophoresis.

Attachment of Template to Beads

In one embodiment, ssDNA libraries that are created according to themethods of the invention are quantitated to calculate the number ofmolecules per unit volume. These molecules are annealed to a solidsupport (bead) that contain oligonucleotide capture primers that arecomplementary to the PCR priming regions of the universal adaptor endsof the ssDNA species. Beads are then transferred to an amplificationprotocol. Clonal populations of single species captured on DNA beads maythen sequenced. In one embodiment, the solid support is a bead,preferably a sepharose bead. As used herein, this bead is referred to asa “DNA capture bead”.

The beads used herein may be of any convenient size and fabricated fromany number of known materials. Example of such materials include:inorganics, natural polymers, and synthetic polymers. Specific examplesof these materials include: cellulose, cellulose derivatives, acrylicresins, glass; silica gels, polystyrene, gelatin, polyvinyl pyrrolidone,co-polymers of vinyl and acrylamide, polystyrene cross-linked withdivinylbenzene or the like (see, Merrifield Biochemistry 1964, 3,1385-1390), polyacrylamides, latex gels, polystyrene, dextran, rubber,silicon, plastics, nitrocellulose, celluloses, natural sponges, silicagels, glass, metals plastic, cellulose, cross-linked dextrans (e.g.,Sephadex™) and agarose gel (Sepharose™) and solid phase supports knownto those of skill in the art. In one embodiment, the diameter of the DNAcapture bead is in the range of 20-70 μm. In a preferred embodiment, thediameter of the DNA capture bead is in a range of 20-50 μm. In a morepreferred embodiment, the diameter of the DNA capture bead is about 30μm.

In one aspect, the invention includes a method for generating a libraryof solid supports comprising: (a) preparing a population of ssDNAtemplates according to the methods disclosed herein; (b) attaching eachDNA template to a solid support such that there is one molecule of DNAper solid support; (c) amplifying the population of single-strandedtemplates such that the amplification generates a clonal population ofeach DNA fragment on each solid support; (d) sequencing clonalpopulations of beads.

In one embodiment, the solid support is a DNA capture bead. In anotherembodiment, the DNA is genomic DNA, cDNA or reverse transcripts of viralRNA. The DNA may be attached to the solid support, for example, via abiotin-streptavidin linkage, a covalent linkage or by complementaryoligonucleotide hybridization. In one embodiment, each DNA template isligated to a set of universal adaptors. In another embodiment, theuniversal adaptor pair comprises a common PCR primer sequence, a commonsequencing primer sequence and a discriminating key sequence.Single-stranded DNAs are isolated that afford unique ends; singlestranded molecules are then attached to a solid support and exposed toamplification techniques for clonal expansion of populations. The DNAmay be amplified by PCR.

In another aspect, the invention provides a library of solid supportsmade by the methods described herein.

The nucleic acid template (e.g., DNA template) prepared by this methodmay be used for many molecular biological procedures, such as linearextension, rolling circle amplification, PCR and sequencing. This methodcan be accomplished in a linkage reaction, for example, by using a highmolar ratio of bead to DNA. Capture of single-stranded DNA moleculeswill follow a poison distribution and will result in a subset of beadswith no DNA attached and a subset of beads with two molecules of DNAattached. In a preferred embodiment, there would be one bead to onemolecule of DNA. In addition, it is possible to include additionalcomponents in the adaptors that may be useful for additionalmanipulations of the isolated library.

2. Nucleic Acid Template Amplification

In order for the nucleic acid template to be sequenced according to themethods of this invention the copy number must be amplified to generatea sufficient number of copies of the template to produce a detectablesignal by the light detection means. Any suitable nucleic acidamplification means may be used.

A number of in vitro nucleic acid amplification techniques have beendescribed. These amplification methodologies may be differentiated intothose methods: (i) which require temperature cycling—polymerase chainreaction (PCR) (see e.g., Saiki, et al., 1995. Science 230: 1350-1354),ligase chain reaction (see e.g., Barany, 1991. Proc. Natl. Acad. Sci.USA 88: 189-193; Barringer, et al., 1990. Gene 89: 117-122) andtranscription-based amplification (see e.g., Kwoh, et al., 1989. Proc.Natl. Acad. Sci. USA 86: 1173-1177) and (ii) isothermal amplificationsystems—self-sustaining, sequence replication (see e.g., Guatelli, etal., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878); the Qβ replicasesystem (see e.g., Lizardi, et al., 1988. BioTechnology 6: 1197-1202);strand displacement amplification Nucleic Acids Res. 1992 Apr. 11;20(7):1691-6; and the methods described in PNAS 1992 Jan. 1;89(1):392-6; and NASBA J Virol Methods. 1991 December; 35(3):273-86.

In one embodiment, isothermal amplification is used. Isothermalamplification also includes rolling circle-based amplification (RCA).RCA is discussed in, e.g., Kool, U.S. Pat. No. 5,714,320 and Lizardi,U.S. Pat. No. 5,854,033; Hatch, et al., 1999. Genet. Anal. Biomol.Engineer. 15: 35-40. The result of the RCA is a single DNA strandextended from the 3′ terminus of the anchor primer (and thus is linkedto the solid support matrix) and including a concatamer containingmultiple copies of the circular template annealed to a primer sequence.Typically, 1,000 to 10,000 or more copies of circular templates, eachhaving a size of, e.g., approximately 30-500, 50-200, or 60-100nucleotides size range, can be obtained with RCA.

The product of RCA amplification following annealing of a circularnucleic acid molecule to an anchor primer is shown schematically in FIG.11A. A circular template nucleic acid 102 is annealed to an anchorprimer 104, which has been linked to a surface 106 at its 5′ end and hasa free 3′ OH available for extension. The circular template nucleic acid102 includes two adapter regions 108 and 110 which are complementary toregions of sequence in the anchor primer 104. Also included in thecircular template nucleic acid 102 is an insert 112 and a region 114homologous to a sequencing primer, which is used in the sequencingreactions described below.

Upon annealing, the free 3′-OH on the anchor primer 104 can be extendedusing sequences within the template nucleic acid 102. The anchor primer102 can be extended along the template multiple times, with eachiteration adding to the sequence extended from the anchor primer asequence complementary to the circular template nucleic acid. Fouriterations, or four rounds of rolling circle replication, are shown inFIG. 1A as the extended anchor primer amplification product 114.Extension of the anchor primer results in an amplification productcovalently or otherwise physically attached to the substrate 106. Anumber of in vitro nucleic acid amplification techniques may be utilizedto extend the anchor primer sequence. The amplification is typicallyperformed in the presence of a polymerase, e.g., a DNA or RNA-directedDNA polymerase, and one, two, three, or four types of nucleotidetriphosphates, and, optionally, auxiliary binding proteins. In general,any polymerase capable of extending a primed 3′-OH group can be used along as it lacks a 3′ to 5′ exonuclease activity. Suitable polymerasesinclude, e.g., the DNA polymerases from Bacillus stearothermophilus,Thermus acquaticus, Pyrococcusfuriosis, Thermococcus litoralis, andThermus thermophilus, bacteriophage T4 and T7, and the E. coli DNApolymerase I Klenow fragment. Suitable RNA-directed DNA polymerasesinclude, e.g., the reverse transcriptase from the Avian MyeloblastosisVirus, the reverse transcriptase from the Moloney Murine Leukemia Virus,and the reverse transcriptase from the Human Immunodeficiency Virus-I.

Additional embodiments of circular templates and anchor primers areshown in more detail in FIG. 11B-11D. FIG. 11B illustrates an annealedopen circle linear substrate that can serve, upon ligation, as atemplate for extension of an anchor primer. A template molecule havingthe sequence 5′—tcg tgt gag gtc tca gca tct tat gta tat tta ctt cta ttctca gtt gcc taa gct gca gcc a—3′ (SEQ ID NO:5) is annealed to an anchorprimer having a biotin linker at its 5′ terminus and the sequence 5′-gacctc aca cga tgg ctg cag ctt—3′ (SEQ ID NO:6). Annealing of the templateresults in juxtaposition of the 5′ and 3′ ends of the template molecule.The 3′OH of the anchor primer can be extended using the circulartemplate.

The use of a circular template and an anchor primer for identificationof single nucleotide polymorphisms is shown in FIG. 11C. Shown is ageneric anchor primer having the sequence 5′—gac ctc aca cga tgg ctg cagctt—3′ (SEQ ID NO:7). The anchor primer anneals to an SNP probe havingthe sequence 5′—ttt ata tgt att cta cga ctc tgg agt gtg cta ccg acg tcgaat ccg ttg act ctt atc ttc a—3′ (SEQ ID NO:8). The SNP probe in turnhybridizes to a region of a SNP-containing region of a gene having thesequence 5′—cta gct cgt aca tat aaa tga aga taa gat cct g—3′ (SEQ IDNO:9). Hybridization of a nucleic acid sequence containing thepolymorphism to the SNP probe complex allows for subsequent ligation andcircularization of the SNP probe. The SNP probe is designed so that its5′ and 3′ termini anneal to the genomic region so as to abut in theregion of the polymorphic site, as is indicated in FIG. 11C. Thecircularized SNP probe can be subsequently extended and sequenced usingthe methods described herein. A nucleic acid lacking the polymorphismdoes not hybridize so as to result in juxtaposition of the 5′ and 3′termini of the SNP probe. In this case, the SNP probe cannot be ligatedto form a circular substrate needed for subsequent extension.

FIG. 11D illustrates the use of a gap oligonucleotide to along with acircular template molecule. An anchor primer having the sequence 5′—gacctc aca cga gta gca tgg ctg cag ctt—3′ (SEQ ID NO:10) is attached to asurface through a biotin linker. A template molecule having the sequence5′—tcg tgt gag gtc tca gca tct tat gta tat tta ctt cta ttc tca gtt gcctaa gct gca gcc a—3′ (SEQ ID NO:11) is annealed to the anchor primer toresult in partially single stranded, or gapped region, in the anchorprimer flanked by a double-stranded region. A gapping molecule havingthe sequence 5′—tgc tac—3′ then anneals to the anchor primer. Ligationof both ends of the gap oligonucleotide to the template molecule resultsin formation of a circular nucleic acid molecule that can act as atemplate for rolling circle amplification.

RCA can occur when the replication of the duplex molecule begins at theorigin. Subsequently, a nick opens one of the strands, and the free3′-terminal hydroxyl moiety generated by the nick is extended by theaction of DNA polymerase. The newly synthesized strand eventuallydisplaces the original parental DNA strand. This aforementioned type ofreplication is known as rolling-circle replication (RCR) because thepoint of replication may be envisaged as “rolling around” the circulartemplate strand and, theoretically, it could continue to do soindefinitely. Additionally, because the newly synthesized DNA strand iscovalently-bound to the original template, the displaced strandpossesses the original genomic sequence (e.g., gene or other sequence ofinterest) at its 5′-terminus. In RCR, the original genomic sequence isfollowed by any number of “replication units” complementary to theoriginal template sequence, wherein each replication unit is synthesizedby continuing revolutions of said original template sequence. Hence,each subsequent revolution displaces the DNA which is synthesized in theprevious replication cycle.

Through the use of the RCA reaction, a strand may be generated whichrepresents many tandem copies of the complement to the circularizedmolecule. For example, RCA has recently been utilized to obtain anisothermal cascade amplification reaction of circularized padlock probesin vitro in order to detect single-copy genes in human genomic DNAsamples (see Lizardi, et al., 1998. Nat. Genet. 19: 225-232). Inaddition, RCA has also been utilized to detect single DNA molecules in asolid phase-based assay, although difficulties arose when this techniquewas applied to in situ hybridization (see Lizardi, et al., 1998. Nat.Genet. 19: 225-232).

If desired, RCA can be performed at elevated temperatures, e.g., attemperatures greater than 37° C., 42° C., 45° C., 50° C., 60° C., or 70°C. In addition, RCA can be performed initially at a lower temperature,e.g., room temperature, and then shifted to an elevated temperature.Elevated temperature RCA is preferably performed with thermostablenucleic acid polymerases and with primers that can anneal stably andwith specificity at elevated temperatures.

RCA can also be performed with non-naturally occurring oligonucleotides,e.g., peptide nucleic acids. Further, RCA can be performed in thepresence of auxiliary proteins such as single-stranded binding proteins.

The development of a method of amplifying short DNA molecules which havebeen immobilized to a solid support, termed RCA has been recentlydescribed in the literature (see e.g., Hatch, et al., 1999. Genet. Anal.Biomol. Engineer. 15: 35-40; Zhang, et al., 1998. Gene 211: 277-85;Baner, et al., 1998. Nucl. Acids Res. 26: 5073-5078; Liu, et al., 1995.J. Am. Chem. Soc. 118: 1587-1594; Fire and Xu, 1995. Proc. Natl. Acad.Sci. USA 92: 4641-4645; Nilsson, et al., 1994. Science 265: 2085-2088).RCA targets specific DNA sequences through hybridization and a DNAligase reaction. The circular product is then subsequently used as atemplate in a rolling circle replication reaction.

Other examples of isothermal amplification systems include, e.g., (i)self-sustaining, sequence replication (see e.g., Guatelli, et al., 1990.Proc. Natl. Acad. Sci. USA 87: 1874-1878), (ii) the Qβ replicase system(see e.g., Lizardi, et al., 1988. BioTechnology 6: 1197-1202), and (iii)nucleic acid sequence-based amplification (NASBA™; see Kievits, et al.,1991. J. Virol. Methods 35: 273-286).

PCR Amplification of Nucleic Acid Templates

In a preferred embodiment, polymerase chain reaction (“PCR”) is used togenerate additional copies of the template nucleic acids. The PCRamplification step may be performed prior to distribution of the nucleicacid templates onto the picotiter plate or may be performed after thenucleic acid templates have been distributed onto the picotiter plate.

Bead Emulsion PCR Amplification

In a preferred embodiment, a PCR amplification step is performed priorto distribution of the nucleic acid templates onto the picotiter plate.

In a particularly preferred embodiment, a novel amplification system,herein termed “bead emulsion amplification” is performed by attaching atemplate nucleic acid (e.g., DNA) to be amplified to a solid support,preferably in the form of a generally spherical bead. A library ofsingle stranded template DNA prepared according to the samplepreparation methods of this invention is an example of one suitablesource of the starting nucleic acid template library to be attached to abead for use in this amplification method.

The bead is linked to a large number of a single primer species (i.e.,primer B in FIG. 6) that is complementary to a region of the templateDNA. Template DNA annealed to the bead bound primer. The beads aresuspended in aqueous reaction mixture and then encapsulated in awater-in-oil emulsion. The emulsion is composed of discrete aqueousphase microdroplets, approximately 60 to 200 um in diameter, enclosed bya thermostable oil phase. Each microdroplet contains, preferably,amplification reaction solution (i.e., the reagents necessary fornucleic acid amplification). An example of an amplification would be aPCR reaction mix (polymerase, salts, dNTPs) and a pair of PCR primers(primer A and primer B). See, FIG. 6A. A subset of the microdropletpopulation also contains the DNA bead comprising the DNA template. Thissubset of microdroplet is the basis for the amplification. Themicrocapsules that are not within this subset have no template DNA andwill not participate in amplification. In one embodiment, theamplification technique is PCR and the PCR primers are present in a 8:1or 16:1 ratio (i.e., 8 or 16 of one primer to 1 of the second primer) toperform asymmetric PCR.

In this overview, the DNA is annealed to an oligonucleotide (primer B)which is immobilized to a bead. During thermocycling (FIG. 6B), the bondbetween the single stranded DNA template and the immobilized B primer onthe bead is broken, releasing the template into the surroundingmicroencapsulated solution. The amplification solution, in this case,the PCR solution, contains addition solution phase primer A and primerB. Solution phase B primers readily bind to the complementary b′ regionof the template as binding kinetics are more rapid for solution phaseprimers than for immobilized primers. In early phase PCR, both A and Bstrands amplify equally well (FIG. 6C).

By midphase PCR (i.e., between cycles 10 and 30) the B primers aredepleted, halting exponential amplification. The reaction then entersasymmetric amplification and the amplicon population becomes dominatedby A strands (FIG. 6D). In late phase PCR (FIG. 6E), after 30 to 40cycles, asymmetric amplification increases the concentration of Astrands in solution. Excess A strands begin to anneal to beadimmobilized B primers. Thermostable polymerases then utilize the Astrand as a template to synthesize an immobilized, bead bound B strandof the amplicon.

In final phase PCR (FIG. 6F), continued thermal cycling forcesadditional annealing to bead bound primers. Solution phase amplificationmay be minimal at this stage but concentration of immobilized B strandsincrease. Then, the emulsion is broken and the immobilized product isrendered single stranded by denaturing (by heat, pH etc.) which removesthe complimentary A strand. The A primers are annealed to the A′ regionof immobilized strand, and immobilized strand is loaded with sequencingenzymes, and any necessary accessory proteins. The beads are thensequenced using recognized pyrophosphate techniques (described, e.g., inU.S. Pat. Nos. 6,274,320, 6,258,568 and 6,210,891, incorporated in totoherein by reference).

Template Design

In a preferred embodiment, the DNA template to be amplified by beademulsion amplification can be a population of DNA such as, for example,a genomic DNA library or a cDNA library. It is preferred that eachmember of the population have a common nucleic acid sequence at thefirst end and a common nucleic acid sequence at a second end. This canbe accomplished, for example, by ligating a first adaptor DNA sequenceto one end and a second adaptor DNA sequence to a second end of the DNApopulation. Many DNA and cDNA libraries, by nature of the cloning vector(e.g., Bluescript, Stratagene, La Jolla, Calif.) fit this description ofhaving a common sequence at a first end and a second common sequence ata second end of each member DNA. The DNA template may be of any sizeamenable to in vitro amplification (including the preferredamplification techniques of PCR and asymmetric PCR). In a preferredembodiment, the DNA template is between about 150 to 750 bp in size,such as, for example about 250 bp in size.

Binding Nucleic Acid Template to Capture Beads

In a first step, a single stranded nucleic acid template to be amplifiedis attached to a capture bead. The nucleic acid template may be attachedto the solid support capture bead in any manner known in the art.Numerous methods exist in the art for attaching DNA to a solid supportsuch as the preferred microscopic bead. According to the presentinvention, covalent chemical attachment of the DNA to the bead can beaccomplished by using standard coupling agents, such as water-solublecarbodiimide, to link the 5′-phosphate on the DNA to amine-coatedcapture beads through a phosphoamidate bond. Another alternative is tofirst couple specific oligonucleotide linkers to the bead using similarchemistry, and to then use DNA ligase to link the DNA to the linker onthe bead. Other linkage chemistries to join the oligonucleotide to thebeads include the use of N-hydroxysuccinamide (NHS) and its derivatives.In such a method, one end of the oligonucleotide may contain a reactivegroup (such as an amide group) which forms a covalent bond with thesolid support, while the other end of the linker contains a secondreactive group that can bond with the oligonucleotide to be immobilized.In a preferred embodiment, the oligonucleotide is bound to the DNAcapture bead by covalent linkage. However, non-covalent linkages, suchas chelation or antigen-antibody complexes, may also be used to join theoligonucleotide to the bead.

Oligonucleotide linkers can be employed which specifically hybridize tounique sequences at the end of the DNA fragment, such as the overlappingend from a restriction enzyme site or the “sticky ends” of bacteriophagelambda based cloning vectors, but blunt-end ligations can also be usedbeneficially. These methods are described in detail in U.S. Pat. No.5,674,743. It is preferred that any method used to immobilize the beadswill continue to bind the immobilized oligonucleotide throughout thesteps in the methods of the invention.

In one embodiment, each capture bead is designed to have a plurality ofnucleic acid primers that recognize (i.e., are complementary to) aportion of the nucleic template, and the nucleic acid template is thushybridized to the capture bead. In the methods described herein, clonalamplification of the template species is desired, so it is preferredthat only one unique nucleic acid template is attached to any onecapture bead.

The beads used herein may be of any convenient size and fabricated fromany number of known materials. Example of such materials include:inorganics, natural polymers, and synthetic polymers. Specific examplesof these materials include: cellulose, cellulose derivatives, acrylicresins, glass, silica gels, polystyrene, gelatin, polyvinyl pyrrolidone,co-polymers of vinyl and acrylamide, polystyrene cross-linked withdivinylbenzene or the like (as described, e.g., in Merrifield,Biochemistry 1964, 3, 1385-1390), polyacrylamides, latex gels,polystyrene, dextran, rubber, silicon, plastics, nitrocellulose, naturalsponges, silica gels, control pore glass, metals, cross-linked dextrans(e.g., Sephadex™) agarose gel (Sepharose™), and solid phase supportsknown to those of skill in the art. In a preferred embodiment, thecapture beads are Sepharose beads approximately 25 to 40 μm in diameter.

Emulsification

Capture beads with attached single strand template nucleic acid areemulsified as a heat stable water-in-oil emulsion. The emulsion may beformed according to any suitable method known in the art. One method ofcreating emulsion is described below but any method for making anemulsion may be used. These methods are known in the art and includeadjuvant methods, counterflow methods, crosscurrent methods, rotatingdrum methods, and membrane methods. Furthermore, the size of themicrocapsules may be adjusted by varying the flow rate and speed of thecomponents. For example, in dropwise addition, the size of the drops andthe total time of delivery may be varied. Preferably, the emulsioncontains a density of bead “microreactors” at a density of about 3,000beads per microliter.

The emulsion is preferably generated by suspending the template-attachedbeads in amplification solution. As used herein, the term “amplificationsolution” means the sufficient mixture of reagents that is necessary toperform amplification of template DNA. One example of an amplificationsolution, a PCR amplification solution, is provided in the Examplesbelow—it will be appreciated that various modifications may be made tothe PCR solution.

In one embodiment, the bead/amplification solution mixture is addeddropwise into a spinning mixture of biocompatible oil (e.g., lightmineral oil, Sigma) and allowed to emulsify. The oil used may besupplemented with one or more biocompatible emulsion stabilizers. Theseemulsion stabilizers may include Atlox 4912, Span 80, and otherrecognized and commercially available suitable stabilizers. Preferably,the droplets formed range in size from 5 micron to 500 microns, morepreferably, from between about 50 to 300 microns, and most preferably,from 100 to 150 microns.

There is no limitation in the size of the microreactors. Themicroreactors should be sufficiently large to encompass sufficientamplification reagents for the degree of amplification required.However, the microreactors should be sufficiently small so that apopulation of microreactors, each containing a member of a DNA library,can be amplified by conventional laboratory equipment (e.g., PCRthermocycling equipment, test tubes, incubators and the like).

With the limitations described above, the optimal size of a microreactormay be between 100 to 200 microns in diameter. Microreactors of thissize would allow amplification of a DNA library comprising about 600,000members in a suspension of microreactors of less than 10 ml in volume.For example, if PCR was the chosen amplification method, 10 mls wouldfit in 96 tubes of a regular thermocycler with 96 tube capacity. In apreferred embodiment, the suspension of 600,000 microreactors would havea volume of less than 1 ml. A suspension of less than 1 ml may beamplified in about 10 tubes of a conventional PCR thermocycler. In amost preferred embodiment, the suspension of 600,000 microreactors wouldhave a volume of less than 0.5 ml.

Amplification

After encapsulation, the template nucleic acid may be amplified by anysuitable method of DNA amplification including transcription-basedamplification systems (Kwoh D. et al., Proc. Natl. Acad. Sci. (U.S.A.)86:1173 (1989); Gingeras T. R. et al., PCT appl. WO 88/10315; Davey, C.et al., European Patent Application Publication No. 329,822; Miller, H.I. et al., PCT appl. WO 89/06700, and “race” (Frohman, M. A., In: PCRProtocols: A Guide to Methods and Applications, Academic Press, NY(1990)) and “one-sided PCR” (Ohara, O. et al., Proc. Natl. Acad. Sci.(U.S.A.) 86.5673-5677 (1989)). Still other less common methods such as“di-oligonucleotide” amplification, isothermal amplification (Walker, G.T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)), androlling circle amplification (reviewed in U.S. Pat. No. 5,714,320), maybe used in the present invention.

In a preferred embodiment, DNA amplification is performed by PCR. PCRaccording to the present invention may be performed by encapsulating thetarget nucleic acid, bound to a bead, with a PCR solution comprising allthe necessary reagents for PCR. Then, PCR may be accomplished byexposing the emulsion to any suitable thermocycling regimen known in theart. In a preferred embodiment, between 30 and 50 cycles, preferablyabout 40 cycles, of amplification are performed. It is desirable, butnot necessary, that following the amplification procedure there be oneor more hybridization and extension cycles following the cycles ofamplification. In a preferred embodiment, between 10 and 30 cycles,preferably about 25 cycles, of hybridization and extension are performed(e.g., as described in the examples). Routinely, the template DNA isamplified until typically at least two million to fifty million copies,preferably about ten million to thirty million copies of the templateDNA are immobilized per bead.

Breaking the Emulsion and Bead Recovery

Following amplification of the template, the emulsion is “broken” (alsoreferred to as “demulsification” in the art). There are many methods ofbreaking an emulsion (see, e.g., U.S. Pat. No. 5,989,892 and referencescited therein) and one of skill in the art would be able to select theproper method. In the present invention, one preferred method ofbreaking the emulsion is to add additional oil to cause the emulsion toseparate into two phases. The oil phase is then removed, and a suitableorganic solvent (e.g., hexanes) is added. After mixing, the oil/organicsolvent phase is removed. This step may be repeated several times.Finally, the aqueous layers above the beads are removed. The beads arethen washed with an organic solvent/annealing buffer mixture (e.g., onesuitable annealing buffer is described in the examples), and then washedagain in annealing buffer. Suitable organic solvents include alcoholssuch as methanol, ethanol and the like.

The amplified template-containing beads may then be resuspended inaqueous solution for use, for example, in a sequencing reactionaccording to known technologies. (See, Sanger, F. et al., Proc. Natl.Acad. Sci. U.S.A. 75, 5463-5467 (1977); Maxam, A. M. & Gilbert, W. ProcNatl Acad Sci USA 74, 560-564 (1977); Ronaghi, M. et al., Science 281,363, 365 (1998); Lysov, I. et al., Dokl Akad Nauk SSSR 303, 1508-1511(1988); Bains W. & Smith G. C. J. TheorBiol 135, 303-307 (1988);Drnanac, R. et al., Genomics 4, 114-128 (1989); Khrapko, K. R. et al.,FEBS Lett 256. 118-122 (1989); Pevzner P. A. J Biomol Struct Dyn 7,63-73 (1989); Southern, E. M. et al., Genomics 13, 1008-1017 (1992).) Ifthe beads are to be used in a pyrophosphate-based sequencing reaction(described, e.g., in U.S. Pat. Nos. 6,274,320, 6,258,568 and 6,210,891,and incorporated in toto herein by reference), then it is necessary toremove the second strand of the PCR product and anneal a sequencingprimer to the single stranded template that is bound to the bead.

Briefly, the second strand is melted away using any number of commonlyknown methods such as NaOH, low ionic (e.g., salt) strength, or heatprocessing. Following this melting step, the beads are pelleted and thesupernatant is discarded. The beads are resuspended in an annealingbuffer, the sequencing primer added, and annealed to the bead-attachedsingle stranded template using a standard annealing cycle.

Purifying the Beads

At this point, the amplified DNA on the bead may be sequenced eitherdirectly on the bead or in a different reaction vessel. In an embodimentof the present invention, the DNA is sequenced directly on the bead bytransferring the bead to a reaction vessel and subjecting the DNA to asequencing reaction (e.g., pyrophosphate or Sanger sequencing).Alternatively, the beads may be isolated and the DNA may be removed fromeach bead and sequenced. In either case, the sequencing steps may beperformed on each individual bead. However, this method, whilecommercially viable and technically feasible, may not be most effectivebecause many of the beads will be negative beads (a bead that does nothave amplified DNA attached). Accordingly, the following optionalprocess may be used for removing beads that contain no nucleic acidtemplate prior to distribution onto the picotiter plate.

A high percentage of the beads may be “negative” (i.e., have noamplified nucleic acid template attached thereto) if the goal of theinitial DNA attachment is to minimize beads with two different copies ofDNA. For useful pyrophosphate sequencing, each bead should containmultiple copies of a single species of DNA. This requirement is mostclosely approached by maximizing the total number of beads with a singlefragment of DNA bound (before amplification). This goal can be achievedby the observation of a mathematical model.

For the general case of “N” number of DNAs randomly distributed among Mnumber of beads, the relative bead population containing any number ofDNAs depends on the ratio of N/M. The fraction of beads containing NDNAs R(N) may be calculated using the Poisson distribution:R(N)=exp—(N/M)X(N/M)^(N) /N! (where X is the multiplication symbol)

The table below shows some calculated values for various N/M (theaverage DNA fragment to bead ratio) and N (the number of fragmentsactually bound to a bead).

N/M 0.1 0.5 1 2 R(0) 0.9 0.61 0.37 0.13 R(1) 0.09 0.3 0.37 0.27 R(N > 1)0.005 0.09 0.26 0.59

In the table the top row denotes the various ratios of N/M. R(0) denotesthe fraction of beads with no DNA, R(1) denotes the fraction of beadswith one DNA attached (before amplification) and R(N>1) denotes thefraction of DNA with more than one DNA attached (before amplification).

The table indicates that the maximum fraction of beads containing asingle DNA fragment is 0.37 (37%) and occurs at a fragment to bead ratioof one. In this mixture, about 63% of the beads is useless forsequencing because they have either no DNA or more than a single speciesof DNA. Additionally, controlling the fragment to bead ratio requirecomplex calculations and variability could produce bead batches with asignificantly smaller fraction of useable beads.

This inefficiency could be significantly ameliorated if beads containingamplicon (originating from the binding of at least one fragment) couldbe separated from those without amplicon (originating from beads with nobound fragments). An amplicon is defined as any nucleic acid moleculesproduced by an in vitro nucleic amplification technique. Binding wouldbe done at low average fragment-to-bead ratios (N/M<1), minimizing theratio of beads with more than one DNA bound. A separation step wouldremove most or all of the beads with no DNA leaving an enrichedpopulation of beads with one species of amplified DNA. These beads maybe applied to any method of sequencing such as, for example,pyrophosphate sequencing. Because the fraction of beads with oneamplicon (N=1) has been enriched, any method of sequencing would be moreefficient.

As an example, with an average fragment to bead ratio of 0.1, 90% of thebeads will have no amplicon, 9% of the beads would be useful with oneamplicon, and 0.5% of the beads will have more than one amplicon. Anenrichment process of the invention will remove the 90% of the zeroamplicon beads leaving a population of beads where the sequenceablefraction (N=1) is:1−(0.005/0.09)=94%.

Dilution of the fragment to bead mixture, along with separation of beadscontaining amplicon can yield an enrichment of 2.5 folds over theoptimal unenriched method. 94%/37% (see table above N/M=1)=2.5. Anadditional benefit of the enrichment procedure of the invention is thatthe ultimate fraction of sequenceable beads is relatively insensitive tovariability in N/M. Thus, complex calculations to derive the optimal N/Mratio are either unnecessary or may be performed to a lower level ofprecision. This will ultimately make the procedure more suitable toperformance by less trained personnel or automation. An additionalbenefit of the procedure is that the zero amplicon beads may be recycledand reused. While recycling is not necessary, it may reduce cost or thetotal bulk of reagents making the method of the invention more suitablefor some purposes such as, for example, portable sampling, remoterobotic sampling and the like. In addition, all the benefits of theprocedure (i.e., less trained personnel, automation, recycling ofreagents) will reduce the cost of the procedure. The procedure isdescribed in more detail below.

The enrichment procedure may be used to treat beads that have beenamplified in the bead emulsion method above. The amplification isdesigned so that each amplified molecule contains the same DNA sequenceat its 3′ end. The nucleotide sequence may be a 20 mer but may be anysequence from 15 bases or more such as 25 bases, 30 bases, 35 bases, or40 bases or longer. Naturally, while longer oligonucleotide ends arefunctional, they are not necessary. This DNA sequence may be introducedat the end of an amplified DNA by one of skill in the art. For example,if PCR is used for amplification of the DNA, the sequence may be part ofone member of the PCR primer pair.

A schematic of the enrichment process is illustrated in FIG. 7. Here,the amplicon-bound bead mixed with 4 empty beads represents thefragment-diluted amplification bead mixture. In step 1, a biotinylatedprimer complementary to the 3′ end of the amplicon is annealed to theamplicon. In step 2, DNA polymerase and the four naturaldeoxynucleotides triphosphates (dNTPs) are added to the bead mix and thebiotinylated primer is extended. This extension is to enhance thebonding between the biotinylated primer and the bead-bound DNA. Thisstep may be omitted if the biotinylated primer—DNA bond is strong (e.g.,in a high ionic environment). In Step 3, streptavidin coated beadssusceptible to attraction by a magnetic field (referred to herein as“magnetic streptavidin beads”) are introduced to the bead mixtures.Magnetic beads are commercially available, for example, from Dynal(M290). The streptavidin capture moieties binds biotins hybridized tothe amplicons, which then specifically fix the amplicon-bound beads tothe magnetic streptavidin beads.

In step 5, a magnetic field (represented by a magnet) is applied nearthe reaction mixture, which causes all the “magnetic streptavidinbeads/amplicon bound bead complexes” to be positioned along one side ofthe tube most proximal to the magnetic field. Magnetic beads withoutamplicon bound beads attached are also expected to be positioned alongthe same side. Beads without amplicons remain in solution. The beadmixture is washed and the beads not immobilized by the magnet (i.e., theempty beads) are removed and discarded. In step 6, the extendedbiotinylated primer strand is separated from the amplicon strand by“melting”—a step that can be accomplished, for example, by heat or achange in pH. The heat may be 60° C. in low salt conditions (e.g., in alow ionic environment such as 0.1×SSC). The change in pH may beaccomplished by the addition of NaOH. The mixture is then washed and thesupernatant, containing the amplicon bound beads, is recovered while thenow unbound magnetic beads are retained by a magnetic field. Theresultant enriched beads may be used for DNA sequencing. It is notedthat the primer on the DNA capture bead may be the same as the primer ofstep 2 above. In this case, annealing of the amplicon-primercomplementary strands (with or without extension) is the source oftarget-capture affinity.

The biotin streptavidin pair could be replaced by a variety ofcapture-target pairs. Two categories are pairs whose binding can besubsequently cleaved and those which bind irreversibly, under conditionsthat are practically achievable. Cleavable pairs include thiol-thiol,Digoxigenin/anti-Digoxigenin, -Captavidin™ if cleavage of thetarget-capture complex is desired.

As described above, step 2 is optional. If step 2 is omitted, it may notbe necessary to separate the magnetic beads from the amplicon boundbeads. The amplicon bound beads, with the magnetic beads attached, maybe used directly for sequencing. If the sequencing were to be performedin a microwell, separation would not be necessary if the amplicon boundbead-magnetic bead complex can fit inside the microwell.

While the use of magnetic capture beads is convenient, capture moietiescan be bound to other surfaces. For example, streptavidin could bechemically bound to a surface, such as, the inner surface of a tube. Inthis case, the amplified bead mixture may be flowed through. Theamplicon bound beads will tend to be retained until “melting” while theempty beads will flow through. This arrangement may be particularlyadvantageous for automating the bead preparation process.

While the embodiments described above is particularly useful, othermethods can be envisioned to separate beads. For example, the capturebeads may be labeled with a fluorescent moiety which would make thetarget-capture bead complex fluorescent. The target capture bead complexmay be separated by flow cytometry or fluorescence cell sorter. Usinglarge capture beads would allow separation by filtering or otherparticle size separation techniques. Since both capture and target beadsare capable of forming complexes with a number of other beads, it ispossible to agglutinate a mass of cross-linked capture-target beads. Thelarge size of the agglutinated mass would make separation possible bysimply washing away the unagglutinated empty beads. The methodsdescribed are described in more detail, for example, in Bauer, J.; J.Chromatography B, 722 (1999) 55-69 and in Brody et al., Applied PhysicsLett. 74 (1999) 144-146.

The DNA capture beads each containing multiple copies of a singlespecies of nucleic acid template prepared according to the above methodare then suitable for distribution onto the picotiter plate.

Nucleic Acid Amplification on the Picotiter Plate

In an alternative embodiment, the nucleic acid template is distributedonto the picotiter plate prior to amplification and then amplified insitu on the picotiter plate. This method is described in detail in theExamples.

3. Sequencing the Nucleic Acid Template

Pyrophosphate sequencing is used according to the methods of thisinvention to sequence the nucleic acid template. This technique is basedon the detection of released pyrophosphate (Ppi) during DNA synthesis.See, e.g., Hyman, 1988. A new method of sequencing DNA. Anal Biochem.174:423-36; Ronaghi, 2001. Pyrosequencing sheds light on DNA sequencing.Genome Res. 11:3-11.

In a cascade of enzymatic reactions, visible light is generatedproportional to the number of incorporated nucleotides. The cascadestarts with a nucleic acid polymerization reaction in which inorganicPpi is released with nucleotide incorporation by polymerase. Thereleased Ppi is converted to ATP by ATP sulfurylase, which provides theenergy to luciferase to oxidize luciferin and generates light. Becausethe added nucleotide is known, the sequence of the template can bedetermined. Solid-phase pyrophosphate sequencing utilizes immobilizedDNA in a three-enzyme system (see Figures). To increase thesignal-to-noise ratio, the natural dATP has been replaced by dATPaS.Typically dATPaS is a mixture of two isomers (Sp and Rp); the use ofpure 2′-deoxyadenosine-5′-O′-(1-thiotriphosphate) Sp-isomer inpyrophosphate sequencing allows substantially longer reads, up todoubling of the read length.

4. Apparatus for Sequencing Nucleic Acids

This invention provides an apparatus for sequencing nucleic acids, whichgenerally comprises one or more reaction chambers for conducting asequencing reaction, means for delivering reactants to and from thereaction chamber(s), and means for detecting a sequencing reactionevent. In another embodiment, the apparatus includes a reagent deliverycuvette containing a plurality of cavities on a planar surface. In apreferred embodiment, the apparatus is connected to at least onecomputer for controlling the individual components of the apparatus andfor storing and/or analyzing the information obtained from detection ofthe sequence reaction event.

The invention also provides one or more reaction chambers are arrangedon an inert substrate material, also referred to herein as a “solidsupport”, that allows for discrete localization of the nucleic acidtemplate and of the reactants in a sequencing reaction in a definedspace, as well as for detection of the sequencing reaction event. Thus,as used herein, the terms “reaction chamber” or “analyte reactionchamber” refer to a localized area on the substrate material thatfacilitates interaction of reactants, e.g., in a nucleic acid sequencingreaction. As discussed more fully below, the sequencing reactionscontemplated by the invention preferably occur on numerous individualnucleic acid samples in tandem, in particular simultaneously sequencingnumerous nucleic acid samples derived from genomic and chromosomalnucleic acid templates (e.g., DNA).

The apparatus of the invention therefore preferably comprises asufficient number of reaction chambers to carry out such numerousindividual sequencing reactions. In one embodiment, there are at least10,000 reaction chambers, preferably at least 50,000 reaction chambers,more preferably greater than 100,000 reaction chambers, even morepreferably greater than 200,000 reaction chambers.

Since the number of simultaneous sequencing reactions is limited by thenumber of reaction chambers, the throughput may be increased byfabricating plates containing increasing densities of wells. The tablebelow shows this progression for a 14×43 mm and 30×60 mm active areas,derived from 25×75 mm and 40×75 mm arrays, respectively.

TABLE Development of higher well count arrays. Pitch Well # of Wells #of Wells (um) Diameter (um) (14 × 43 mm) (30 × 60 mm) 50 44 275K 800K 4338 375K 1.2 M 35 31 575K 1.6 M 25 22 1.1 M 3.2 M

The reaction chambers on the array typically take the form of a cavityor well in the substrate material, having a width and depth, into whichreactants can be deposited. Typically the nucleic acid template isdistributed into the reaction chamber on one or more solid supports orbeads; the reactants are in a medium which facilitates the reaction andwhich flows through the reaction chamber. When formed as cavities orwells, the chambers are preferably of sufficient dimension and order toallow for (i) the introduction of the necessary reactants into thechambers, (ii) reactions to take place within the chamber and (iii)inhibition of mixing of reactants between chambers. The shape of thewell or cavity is preferably circular or cylindrical, but can bemultisided so as to approximate a circular or cylindrical shape. In onepreferred embodiment, the shape of the well or cavity is substantiallyhexagonal. The cavity can have a smooth wall surface. In an additionalembodiment, the cavity can have at least one irregular wall surface. Thecavities can have a planar bottom or a concave bottom.

The reaction chambers can be spaced between 5 μm and 200 μm apart.Spacing is determined by measuring the center-to-center distance betweentwo adjacent reaction chambers. Typically, the reaction chambers can bespaced between 10 μm and 150 μm apart, preferably between 20 μm and 100μm apart, most preferably between 40 and 60 μm apart. In one embodiment,the reaction chambers have a width (diameter) in one dimension ofbetween 0.3 μm and 100 μm, more preferably between 20 μm and 70 μm andmost preferably about 30 and 50 μm. The depth of the reaction chambersare preferably between 10 μm and 100 μm, preferably between 20 μm and 60μm. Alternatively, the reaction chambers may have a depth that isbetween 0.25 and 5 times the width in one dimension of the reactionchamber or, in another embodiment, between 0.3 and 1 times the width inone dimension of the reaction chamber.

In a preferred embodiment, the array is fashioned from a sliced fiberoptic bundle (i.e., a bundle of fused fiber optic cables) and thereaction chambers are formed by etching one surface of the fiber opticreactor array. The cavities can also be formed in the substrate viaetching, molding or micromachining.

Each cavity or reaction chamber typically has a depth of between 10 μmand 100 μm; alternatively, the depth is between 0.25 and 5 times thesize of the width of the cavity, preferably between 0.3 and 1 times thesize of the width of the cavity.

In one embodiment, the arrays described herein typically include aplanar top surface and a planar bottom surface, which is opticallyconductive such that optical signals from the reaction chambers can bedetected through the bottom planar surface. In these arrays, typicallythe distance between the top surface and the bottom surface is nogreater than 10 cm, preferably no greater than 2 cm, and usually between0.5 mm to 5 mm, most preferably about 2 mm.

In a particularly preferred embodiment, the solid support is termed apicotiterplate, with reaction chambers having a center to center spacingof about 43 μm to 50 μm, a well diameter of between 38 μm to 44 μm, anda well volume of between 10 to 150 μL, preferably between 20 to 90 μL,more preferably between 40 to 85 μL, and most preferably about 75 μL.

In one embodiment, each cavity or reaction chamber of the array containsreagents for analyzing a nucleic acid or protein. Typically thosereaction chambers that contain a nucleic acid (not all reaction chambersin the array are required to) contain only a single species of nucleicacid (i.e., a single sequence that is of interest). There may be asingle copy of this species of nucleic acid in any particular reactionchamber, or there may be multiple copies. It is generally preferred thata reaction chamber contain at least 100,000 copies of the nucleic acidtemplate sequence, preferably at least 1,000,000 copies, and morepreferably between 2,000,000 to 20,000,000 copies, and most preferablybetween 5,000,000 to 15,000,000 copies of the nucleic acid. Theordinarily skilled artisan will appreciate that changes in the number ofcopies of a nucleic acid species in any one reaction chamber will affectthe number of photons generated in a pyrosequencing reaction, and can beroutinely adjusted to provide more or less photon signal as is required.In one embodiment the nucleic acid species is amplified to provide thedesired number of copies using PCR, RCA, ligase chain reaction, otherisothermal amplification, or other conventional means of nucleic acidamplification. In one embodiment, the nucleic acid is single stranded.

Solid Support Material

Any material can be used as the solid support material, as long as thesurface allows for stable attachment of the primers and detection ofnucleic acid sequences. The solid support material can be planar or canbe cavitated, e.g., in a cavitated terminus of a fiber optic or in amicrowell etched, molded, or otherwise micromachined into the planarsurface, e.g. using techniques commonly used in the construction ofmicroelectromechanical systems. See e.g., Rai-Choudhury, HANDBOOK OFMICROLITHOGRAPHY, MICROMACHINING, AND MICROFABRICATION, VOLUME I:MICROLITHOGRAPHY, Volume PM39, SPIE Press (1997); Madou, CRC Press(1997), Aoki, Biotech. Histochem. 67: 98-9 (1992); Kane et al.,Biomaterials. 20: 2363-76 (1999); Deng et al., Anal. Chem. 72:3176-80(2000); Zhu et al., Nat. Genet. 26:283-9 (2000). In some embodiments,the solid support is optically transparent, e.g., glass.

An array of attachment sites on an optically transparent solid supportcan be constructed using lithographic techniques commonly used in theconstruction of electronic integrated circuits as described in, e.g.,techniques for attachment described in U.S. Pat. Nos. 5,143,854,5,445,934, 5,744,305, and 5,800,992; Chee et al., Science 274: 610-614(1996); Fodor et al., Nature 364: 555-556 (1993); Fodor et al., Science251: 767-773 (1991); Gushin, et al., Anal. Biochem. 250: 203-211 (1997);Kinosita et al., Cell 93: 21-24 (1998); Kato-Yamada et al., J. Biol.Chem. 273: 19375-19377 (1998); and Yasuda et al., Cell 93: 1117-1124(1998). Photolithography and electron beam lithography sensitize thesolid support or substrate with a linking group that allows attachmentof a modified biomolecule (e.g., proteins or nucleic acids). See e.g.,Service, Science 283: 27-28 (1999); Rai-Choudhury, HANDBOOK OFMICROLITHOGRAPHY, MICROMACHINING, AND MICROFABRICATION, VOLUME I:MICROLITHOGRAPHY, Volume PM39, SPIE Press (1997). Alternatively, anarray of sensitized sites can be generated using thin-film technology asdescribed in Zasadzinski et al., Science 263: 1726-1733 (1994).

The substrate material is preferably made of a material that facilitatesdetection of the reaction event. For example, in a typical sequencingreaction, binding of a dNTP to a sample nucleic acid to be sequenced canbe monitored by detection of photons generated by enzyme action onphosphate liberated in the sequencing reaction. Thus, having thesubstrate material made of a transparent or light conductive materialfacilitates detection of the photons.

In some embodiments, the solid support can be coupled to a bundle ofoptical fibers that are used to detect and transmit the light product.The total number of optical fibers within the bundle may be varied so asto match the number of individual reaction chambers in the arrayutilized in the sequencing reaction. The number of optical fibersincorporated into the bundle is designed to match the resolution of adetection device so as to allow 1:1 imaging. The overall sizes of thebundles are chosen so as to optimize the usable area of the detectiondevice while maintaining desirable reagent (flow) characteristics in thereaction chamber. Thus, for a 4096×4096 pixel CCD (charge-coupleddevice) array with 15 μm pixels, the fiber bundle is chosen to beapproximately 60 mm×60 mm or to have a diameter of approximately 90 mm.The desired number of optical fibers are initially fused into a bundleor optical fiber array, the terminus of which can then be cut andpolished so as to form a “wafer” of the required thickness (e.g., 1.5mm). The resulting optical fiber wafers possess similar handlingproperties to that of a plane of glass. The individual fibers can be anysize diameter (e.g., 3 μm to 100 μm).

In some embodiments two fiber optic bundles are used: a first bundle isattached directly to the detection device (also referred to herein asthe fiber bundle or connector) and a second bundle is used as thereaction chamber substrate (the wafer or substrate). In this case thetwo are placed in direct contact, optionally with the use of opticalcoupling fluid, in order to image the reaction centers onto thedetection device. If a CCD is used as the detection device, the wafercould be slightly larger in order to maximize the use of the CCD area,or slightly smaller in order to match the format of a typical microscopeslide—25 mm×75 mm. The diameters of the individual fibers within thebundles are chosen so as to maximize the probability that a singlereaction will be imaged onto a single pixel in the detection device,within the constraints of the state of the art. Exemplary diameters are6-8 μm for the fiber bundle and 6-50 μm for the wafer, though anydiameter in the range 3-100 μm can be used. Fiber bundles can beobtained commercially from CCD camera manufacturers. For example, thewafer can be obtained from Incom, Inc. (Charlton, Mass.) and cut andpolished from a large fusion of fiber optics, typically being 2 mmthick, though possibly being 0.5 to 5 mm thick. The wafer has handlingproperties similar to a pane of glass or a glass microscope slide.

Reaction chambers can be formed in the substrate made from fiber opticmaterial. The surface of the optical fiber is cavitated by treating thetermini of a bundle of fibers, e.g., with acid, to form an indentationin the fiber optic material. Thus, in one embodiment cavities are formedfrom a fiber optic bundle, preferably cavities can be formed by etchingone end of the fiber optic bundle. Each cavitated surface can form areaction chamber. Such arrays are referred to herein as fiber opticreactor arrays or FORA. The indentation ranges in depth fromapproximately one-half the diameter of an individual optical fiber up totwo to three times the diameter of the fiber. Cavities can be introducedinto the termini of the fibers by placing one side of the optical fiberwafer into an acid bath for a variable amount of time. The amount oftime can vary depending upon the overall depth of the reaction cavitydesired (see e.g., Walt, et al., 1996. Anal. Chem. 70: 1888). A widechannel cavity can have uniform flow velocity dimensions ofapproximately 14 mm×43 mm. Thus, with this approximate dimension and atapproximately 4.82×10⁻⁴ cavities/um² density, the apparatus can haveapproximately 290,000 fluidically accessible cavities. Several methodsare known in the art for attaching molecules (and detecting the attachedmolecules) in the cavities etched in the ends of fiber optic bundles.See, e.g., Michael, et al., Anal. Chem. 70: 1242-1248 (1998); Ferguson,et al., Nature Biotechnology 14: 1681-1684 (1996); Healey and Walt,Anal. Chem. 69: 2213-2216 (1997). A pattern of reactive sites can alsobe created in the microwell, using photolithographic techniques similarto those used in the generation of a pattern of reaction pads on aplanar support. See, Healey, et al., Science 269: 1078-1080 (1995);Munkholm and Walt, Anal. Chem. 58: 1427-1430 (1986), and Bronk, et al.,Anal. Chem. 67: 2750-2757 (1995).

The opposing side of the optical fiber wafer (i.e., the non-etched side)is typically highly polished so as to allow optical-coupling (e.g., byimmersion oil or other optical coupling fluids) to a second, opticalfiber bundle. This second optical fiber bundle exactly matches thediameter of the optical wafer containing the reaction chambers, andserve to act as a conduit for the transmission of light product to theattached detection device, such as a CCD imaging system or camera.

In one preferred embodiment, the fiber optic wafer is thoroughlycleaned, e.g by serial washes in 15% H₂O₂/15% NH₄OH volume:volume inaqueous solution, then six deionized water rinses, then 0.5M EDTA, thensix deionized water, then 15% H₂O₂/15% NH₄OH, then six deionized water(one-half hour incubations in each wash).

The surface of the fiber optic wafer is preferably coated to facilitateits use in the sequencing reactions. A coated surface is preferablyoptically transparent, allows for easy attachment of proteins andnucleic acids, and does not negatively affect the activity ofimmobilized proteins. In addition, the surface preferably minimizesnon-specific absorption of macromolecules and increases the stability oflinked macromolecules (e.g., attached nucleic acids and proteins).

Suitable materials for coating the array include, e.g., plastic (e.g.polystyrene). The plastic can be preferably spin-coated or sputtered(0.1 μm thickness). Other materials for coating the array include goldlayers, e.g. 24 karat gold, 0.1 μm thickness, with adsorbedself-assembling monolayers of long chain thiol alkanes. Biotin is thencoupled covalently to the surface and saturated with a biotin-bindingprotein (e.g. streptavidin or avidin).

Coating materials can additionally include those systems used to attachan anchor primer to a substrate. Organosilane reagents, which allow fordirect covalent coupling of proteins via amino, sulfhydryl or carboxylgroups, can also be used to coat the array. Additional coatingsubstances include photoreactive linkers, e.g. photobiotin, (Amos etal., “Biomaterial Surface Modification Using Photochemical CouplingTechnology,” in Encyclopedic Handbook of Biomaterials andBioengineering, Part A: Materials, Wise et al. (eds.), New York, MarcelDekker, pp. 895926, 1995).

Additional coating materials include hydrophilic polymer gels(polyacrylamide, polysaccharides), which preferably polymerize directlyon the surface or polymer chains covalently attached post polymerization(Hjerten, J. Chromatogr. 347,191 (1985); Novotny, Anal. Chem. 62,2478(1990), as well as pluronic polymers (triblock copolymers, e.g.PPO-PEO-PPO, also known as F-108), specifically adsorbed to eitherpolystyrene or silanized glass surfaces (Ho et al., Langmuir 14:3889-94,1998), as well as passively adsorbed layers of biotin-binding proteins.The surface can also be coated with an epoxide which allows the couplingof reagents via an amine linkage.

In addition, any of the above materials can be derivatized with one ormore functional groups, commonly known in the art for the immobilizationof enzymes and nucleotides, e.g. metal chelating groups (e.g. nitrilotriacetic acid, iminodiacetic acid, pentadentate chelator), which willbind 6×His-tagged proteins and nucleic acids.

Surface coatings can be used that increase the number of availablebinding sites for subsequent treatments, e.g. attachment of enzymes(discussed later), beyond the theoretical binding capacity of a 2Dsurface.

In a preferred embodiment, the individual optical fibers utilized togenerate the fused optical fiber bundle/wafer are larger in diameter(i.e., 6 μm to 12 μm) than those utilized in the optical imaging system(i.e., 3 μm). Thus, several of the optical imaging fibers can beutilized to image a single reaction site.

In a particularly preferred embodiment, the sample cartridge for nucleicacid template sequencing, termed the ‘PicoTiter plate’ is formed from acommercial fiber optics faceplate, acid-etched to yield well structures.Each optic fiber core is about 44 microns in diameter, with a 2-3 microncladding, each well formed by acid etching to form a reaction wellvolume of about 65 μL to 85 μL, most preferably about 75 μL. The use ofetched wells on a fiber optics faceplate surface serves a threefoldpurpose; i) delayed diffusion of the luminescence from emitting light ina different region of the array, ii) isolation of reaction chambers(“test-tubes”) that contain the amplified template molecules, and iii)very efficient, high numerical aperture optical coupling to the CCD.Finally, the larger the amount of sequencing template immobilized withina well, the more optical signal one is able to achieve.

Delivery Means

An example of the means for delivering reactants to the reaction chamberis the perfusion chamber of the present invention is illustrated in FIG.13. The perfusion chamber includes a sealed compartment with transparentupper and lower side. It is designed to allow flow of solution over thesurface of the substrate surface and to allow for fast exchange ofreagents. Thus, it is suitable for carrying out, for example, thepyrophosphate sequencing reactions. The shape and dimensions of thechamber can be adjusted to optimize reagent exchange to include bulkflow exchange, diffusive exchange, or both in either a laminar flow or aturbulent flow regime.

The perfusion chamber is preferably detached from the imaging systemwhile it is being prepared and only placed on the imaging system whensequencing analysis is performed. In one embodiment, the solid support(i.e., a DNA chip or glass slide) is held in place by a metal or plastichousing, which may be assembled and disassembled to allow replacement ofsaid solid support. The lower side of the solid support of the perfusionchamber carries the reaction chamber array and, with a traditionaloptical-based focal system, a high numerical aperture objective lens isused to focus the image of the reaction center array onto the CCDimaging system.

Many samples can thereby be analyzed in parallel. Using the method ofthe invention, many nucleic acid templates may be analyzed in this wasby allowing the solution containing the enzymes and one nucleotide toflow over the surface and then detecting the signal produced for eachsample. This procedure can then be repeated. Alternatively, severaldifferent oligonucleotides complementary to the template may bedistributed over the surface followed by hybridization of the template.Incorporation of deoxynucleotides or dideoxynucleotides may be monitoredfor each oligonucleotide by the signal produced using the variousoligonucleotides as primer. By combining the signals from differentareas of the surface, sequence-based analyses may be performed by fourcycles of polymerase reactions using the various dideoxynucleotides.

When the support is in the form of a cavitated array, e.g., in thetermini of a picotiter plate or other array of microwells, suitabledelivery means for reagents include flowing and washing and also, e.g.,flowing, spraying, electrospraying, ink jet delivery, stamping,ultrasonic atomization (Sonotek Corp., Milton, N.Y) and rolling. Whenspraying is used, reagents may be delivered to the picotiter plate in ahomogeneous thin layer produced by industrial type spraying nozzles(Spraying Systems, Co., Wheaton, Ill.) or atomizers used in thin layerchromatography (TLC), such as CAMAG TLC Sprayer (Camag Scientific Inc.,Wilmington, N.C.). These sprayers atomize reagents into aerosol sprayparticles in the size range of 0.3 to 10 μm.

Successive reagent delivery steps are preferably separated by wash stepsusing techniques commonly known in the art. These washes can beperformed, e.g., using the above described methods, including high-flowsprayers or by a liquid flow over the picotiter plate or microwell arraysurface. The washes can occur in any time period after the startingmaterial has reacted with the reagent to form a product in each reactionchamber but before the reagent delivered to any one reaction chamber hasdiffused out of that reaction chamber into any other reaction chamber.In one embodiment, any one reaction chamber is independent of theproduct formed in any other reaction chamber, but is generated using oneor more common reagents.

An embodiment of a complete apparatus is illustrated in FIG. 12. Theapparatus includes an inlet conduit 200 in communication with adetachable perfusion chamber 226. The inlet conduit 200 allows for entryof sequencing reagents via a plurality of tubes 202-212, which are eachin communication with a plurality of sequencing dispensing reagentvessels 214-224.

Reagents are introduced through the conduit 200 into the perfusionchamber 226 using either a pressurized system or pumps to drive positiveflow. Typically, the reagent flow rates are from 0.05 to 50 ml/minute(e.g., 1 to 50 ml/minute) with volumes from 0.100 ml to continuous flow(for washing). Valves are under computer control to allow cycling ofnucleotides and wash reagents. Sequencing reagents, e.g., polymerase canbe either pre-mixed with nucleotides or added in stream. A manifoldbrings all six tubes 202-212 together into one for feeding the perfusionchamber. Thus several reagent delivery ports allow access to theperfusion chamber. For example, one of the ports may be utilized toallow the input of the aqueous sequencing reagents, while another portallows these reagents (and any reaction products) to be withdrawn fromthe perfusion chamber.

In a preferred embodiment, one or more reagents are delivered to anarray immobilized or attached to a population of mobile solid supports,e.g., a bead or microsphere. The bead or microsphere need not bespherical, irregular shaped beads may be used. They are typicallyconstructed from numerous substances, e.g., plastic, glass or ceramicand bead sizes ranging from nanometers to millimeters depending on thewidth of the reaction chamber. Various bead chemistries can be usede.g., methylstyrene, polystyrene, acrylic polymer, latex, paramagnetic,thoria sol, carbon graphite and titanium dioxide. The construction orchemistry of the bead can be chosen to facilitate the attachment of thedesired reagent.

In another embodiment, the bioactive agents are synthesized first, andthen covalently attached to the beads. As is appreciated by someoneskilled in the art, this will be done depending on the composition ofthe bioactive agents and the beads. The functionalization of solidsupport surfaces such as certain polymers with chemically reactivegroups such as thiols, amines, carboxyls, etc. is generally known in theart.

In a preferred embodiment, the nucleic acid template is delivered to thepicotiter plate on beads. The luciferase and sulfurylase enzymes arelikewise delivered to each well on beads (see Figure), as is the DNApolymerase. It is noted that the one or more of the nucleic acidtemplate, luciferase and sulfurylase may be delivered each on separatebeads, or together on the same bead. To allow sequencing DNA at raisedtemperatures, we have cloned and modified the thermostable sulfurylasefrom Bacillus steareothermophilus. We have also cloned and modifiedseveral luciferase enzymes for solid-phase enzyme activity, including P.pennsylvanica and P. pyralis. The P. pyralis luciferase is used in apreferred embodiment.

“Blank” beads may be used that have surface chemistries that facilitatethe attachment of the desired functionality by the user. Additionalexamples of these surface chemistries for blank beads include, but arenot limited to, amino groups including aliphatic and aromatic amines,carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazide,hydroxyl groups, sulfonates and sulfates.

These functional groups can be used to add any number of differentcandidate agents to the beads, generally using known chemistries. Forexample, candidate agents containing carbohydrates may be attached to anamino-functionalized support; the aldehyde of the carbohydrate is madeusing standard techniques, and then the aldehyde is reacted with anamino group on the surface. In an alternative embodiment, a sulfhydryllinker may be used. There are a number of sulfhydryl reactive linkersknown in the art such as SPDP, maleimides, α-haloacetyls, and pyridyldisulfides (see for example the 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated here byreference) which can be used to attach cysteine containing proteinaceousagents to the support. Alternatively, an amino group on the candidateagent may be used for attachment to an amino group on the surface. Forexample, a large number of stable bifunctional groups are well known inthe art, including homobifunctional and heterobifunctional linkers (seePierce Catalog and Handbook, pages 155-200). In an additionalembodiment, carboxyl groups (either from the surface or from thecandidate agent) may be derivatized using well known linkers (see Piercecatalog). For example, carbodiimides activate carboxyl groups for attackby good nucleophiles such as amines (see Torchilin et al., Critical Rev.Thereapeutic Drug Carrier Systems, 7(4):275-308 (1991)). Proteinaceouscandidate agents may also be attached using other techniques known inthe art, for example for the attachment of antibodies to polymers; seeSlinkin et al., Bioconj. Chem. 2:342-348 (1991); Torchilin et al.,supra; Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al.,Cancer Res. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem.5:220-235 (1994). It should be understood that the candidate agents maybe attached in a variety of ways, including those listed above.Preferably, the manner of attachment does not significantly alter thefunctionality of the candidate agent; that is, the candidate agentshould be attached in such a flexible manner as to allow its interactionwith a target.

Specific techniques for immobilizing enzymes on beads are known in theprior art. In one case, NH₂ surface chemistry beads are used. Surfaceactivation is achieved with a 2.5% glutaraldehyde in phosphate bufferedsaline (10 mM) providing a pH of 6.9 (138 mM NaCl, 2.7 mM KCl). Thismixture is stirred on a stir bed for approximately 2 hours at roomtemperature. The beads are then rinsed with ultrapure water plus 0.01%Tween 20 (surfactant)—0.02%, and rinsed again with a pH 7.7 PBS plus0.01% tween 20. Finally, the enzyme is added to the solution, preferablyafter being prefiltered using a 0.45 μm a micon micropure filter.

The population of mobile solid supports are disposed in the reactionchambers. In some embodiments, 5% to 20% of the reaction chambers canhave a mobile solid support with at least one reagent immobilizedthereon, 20% to 60% of the reaction chambers can have a mobile solidsupport with at least one reagent immobilized thereon or 50% to 100% ofthe reaction chambers can have a mobile solid support with at least onereagent immobilized thereon. Preferably, at least one reaction chamberhas a mobile solid support having at least one reagent immobilizedthereon and the reagent is suitable for use in a nucleic acid sequencingreaction.

In some embodiments, the reagent immobilized to the mobile solid supportcan be a polypeptide with sulfurylase activity, a polypeptide withluciferase activity or a chimeric polypeptide having both sulfurylaseand luciferase activity. In one embodiment, it can be a ATP sulfurylaseand luciferase fusion protein. Since the product of the sulfurylasereaction is consumed by luciferase, proximity between these two enzymesmay be achieved by covalently linking the two enzymes in the form of afusion protein. This invention would be useful not only in substratechanneling but also in reducing production costs and potentiallydoubling the number of binding sites on streptavidin-coated beads.

In another embodiment, the sulfurylase is a thermostable ATPsulfurylase. In a preferred embodiment, the thermostable sulfurylase isactive at temperatures above ambient (to at least 50° C.). In oneembodiment, the ATP sulfurylase is from a thermophile. In an additionalembodiment, the mobile solid support can have a first reagent and asecond reagent immobilized thereon, the first reagent is a polypeptidewith sulfurylase activity and the second reagent is a polypeptide withluciferase activity.

In another embodiment, the reagent immobilized to the mobile solidsupport can be a nucleic acid; preferably the nucleic acid is a singlestranded concatamer. In a preferred embodiment, the nucleic acid can beused for sequencing a nucleic acid, e.g., a pyrosequencing reaction.

The invention also provides a method for detecting or quantifying ATPactivity using a mobile solid support; preferably the ATP can bedetected or quantified as part of a nucleic acid sequencing reaction.

A picotiter plate that has been “carpeted” with mobile solid supportswith either nucleic acid or reagent enzymes attached thereto is shown asFIG. 15.

5. Methods of Sequencing Nucleic Acids

Pyrophosphate-based sequencing is then performed. The sample DNAsequence and the extension primer are then subjected to a polymerasereaction in the presence of a nucleotide triphosphate whereby thenucleotide triphosphate will only become incorporated and releasepyrophosphate (PPi) if it is complementary to the base in the targetposition, the nucleotide triphosphate being added either to separatealiquots of sample-primer mixture or successively to the samesample-primer mixture. The release of PPi is then detected to indicatewhich nucleotide is incorporated.

In one embodiment, a region of the sequence product is determined byannealing a sequencing primer to a region of the template nucleic acid,and then contacting the sequencing primer with a DNA polymerase and aknown nucleotide triphosphate, i.e., dATP, dCTP, dGTP, dTTP, or ananalog of one of these nucleotides. The sequence can be determined bydetecting a sequence reaction byproduct, as is described below.

The sequence primer can be any length or base composition, as long as itis capable of specifically annealing to a region of the amplifiednucleic acid template. No particular structure for the sequencing primeris required so long as it is able to specifically prime a region on theamplified template nucleic acid. Preferably, the sequencing primer iscomplementary to a region of the template that is between the sequenceto be characterized and the sequence hybridizable to the anchor primer.The sequencing primer is extended with the DNA polymerase to form asequence product. The extension is performed in the presence of one ormore types of nucleotide triphosphates, and if desired, auxiliarybinding proteins.

Incorporation of the dNTP is preferably determined by assaying for thepresence of a sequencing byproduct. In a preferred embodiment, thenucleotide sequence of the sequencing product is determined by measuringinorganic pyrophosphate (PPi) liberated from a nucleotide triphosphate(dNTP) as the dNMP is incorporated into an extended sequence primer.This method of sequencing, termed Pyrosequencing™ technology(PyroSequencing AB, Stockholm, Sweden) can be performed in solution(liquid phase) or as a solid phase technique. PPi-based sequencingmethods are described generally in, e.g., WO9813523A1, Ronaghi, et al.,1996. Anal. Biochem. 242: 84-89, Ronaghi, et al., 1998. Science 281:363-365 (1998) and USSN 2001/0024790. These disclosures of PPisequencing are incorporated herein in their entirety, by reference. Seealso, e.g., U.S. Pat. Nos. 6,210,891 and 6,258,568, each fullyincorporated herein by reference.

Pyrophosphate released under these conditions can be detectedenzymatically (e.g., by the generation of light in theluciferase-luciferin reaction). Such methods enable a nucleotide to beidentified in a given target position, and the DNA to be sequencedsimply and rapidly while avoiding the need for electrophoresis and theuse of potentially dangerous radiolabels.

PPi can be detected by a number of different methodologies, and variousenzymatic methods have been previously described (see e.g., Reeves, etal., 1969. Anal. Biochem. 28: 282-287; Guillory, et al., 1971. Anal.Biochem. 39: 170-180; Johnson, et al., 1968. Anal. Biochem. 15: 273;Cook, et al., 1978. Anal. Biochem. 91: 557-565; and Drake, et al., 1979.Anal. Biochem. 94: 117-120).

PPi liberated as a result of incorporation of a dNTP by a polymerase canbe converted to ATP using, e.g., an ATP sulfurylase. This enzyme hasbeen identified as being involved in sulfur metabolism. Sulfur, in bothreduced and oxidized forms, is an essential mineral nutrient for plantand animal growth (see e.g., Schmidt and Jager, 1992. Ann. Rev. PlantPhysiol. Plant Mol. Biol. 43: 325-349). In both plants andmicroorganisms, active uptake of sulfate is followed by reduction tosulfide. As sulfate has a very low oxidation/reduction potentialrelative to available cellular reductants, the primary step inassimilation requires its activation via an ATP-dependent reaction (seee.g., Leyh, 1993. Crit. Rev. Biochem. Mol. Biol. 28: 515-542). ATPsulfurylase (ATP: sulfate adenylyltransferase; EC 2.7.7.4) catalyzes theinitial reaction in the metabolism of inorganic sulfate (SO₄ ⁻²); seee.g., Robbins and Lipmann, 1958. J. Biol. Chem. 233: 686-690; Hawes andNicholas, 1973. Biochem. J. 133: 541-550). In this reaction SO₄ ⁻² isactivated to adenosine 5′-phosphosulfate (APS).

ATP sulfurylase has been highly purified from several sources, such asSaccharomyces cerevisiae (see e.g., Hawes and Nicholas, 1973. Biochem.J. 133: 541-550); Penicillium chrysogenum (see e.g., Renosto, et al.,1990. J. Biol. Chem. 265: 10300-10308); rat liver (see e.g., Yu, et al.,1989. Arch. Biochem. Biophys. 269: 165-174); and plants (see e.g., Shawand Anderson, 1972. Biochem. J. 127: 237-247; Osslund, et al., 1982.Plant Physiol. 70: 39-45). Furthermore, ATP sulfurylase genes have beencloned from prokaryotes (see e.g., Leyh, et al., 1992. J. Biol. Chem.267: 10405-10410; Schwedock and Long, 1989. Mol. Plant. MicrobeInteraction 2: 181-194; Laue and Nelson, 1994. J. Bacteriol. 176:3723-3729); eukaryotes (see e.g., Cherest, et al., 1987. Mol. Gen.Genet. 210: 307-313; Mountain and Korch, 1991. Yeast 7: 873-880; Foster,et al., 1994. J. Biol. Chem. 269: 19777-19786); plants (see e.g.,Leustek, et al., 1994. Plant Physiol. 105: 897-90216); and animals (seee.g., Li, et al., 1995. J. Biol. Chem. 270: 29453-29459). The enzyme isa homo-oligomer or heterodimer, depending upon the specific source (seee.g., Leyh and Suo, 1992. J. Biol. Chem. 267: 542-545).

In some embodiments, a thermostable sulfurylase is used. Thermostablesulfurylases can be obtained from, e.g., Archaeoglobus or Pyrococcusspp. Sequences of thermostable sulfurylases are available at databaseAcc. No. 028606, Acc. No. Q9YCR4, and Acc. No. P56863.

ATP sulfurylase has been used for many different applications, forexample, bioluminometric detection of ADP at high concentrations of ATP(see e.g., Schultz, et al., 1993. Anal. Biochem. 215: 302-304);continuous monitoring of DNA polymerase activity (see e.g., Nyrbn, 1987.Anal. Biochem. 167: 235-238); and DNA sequencing (see e.g., Ronaghi, etal., 1996. Anal. Biochem. 242: 84-89; Ronaghi, et al., 1998. Science281: 363-365; Ronaghi, et al., 1998. Anal. Biochem. 267: 65-71).

Several assays have been developed for detection of the forward ATPsulfurylase reaction. The colorimetric molybdolysis assay is based onphosphate detection (see e.g., Wilson and Bandurski, 1958. J. Biol.Chem. 233: 975-981), whereas the continuous spectrophotometricmolybdolysis assay is based upon the detection of NADH oxidation (seee.g., Seubert, et al., 1983. Arch. Biochem. Biophys. 225: 679-691;Seubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523). The laterassay requires the presence of several detection enzymes. In addition,several radioactive assays have also been described in the literature(see e.g., Daley, et al., 1986. Anal. Biochem. 157: 385-395). Forexample, one assay is based upon the detection of ³²PPi released from³²P-labeled ATP (see e.g., Seubert, et al., 1985. Arch. Biochem.Biophys. 240: 509-523) and another on the incorporation of ³⁵S into[³⁵S]-labeled APS (this assay also requires purified APS kinase as acoupling enzyme; see e.g., Seubert, et al., 1983. Arch. Biochem.Biophys. 225: 679-691); and a third reaction depends upon the release of35SO₄ ⁻² from [³⁵S]-labeled APS (see e.g., Daley, et al., 1986. Anal.Biochem. 157: 385-395).

For detection of the reversed ATP sulfurylase reaction a continuousspectrophotometric assay (see e.g., Segel, et al., 1987. MethodsEnzymol. 143: 334-349); a bioluminometric assay (see e.g. Balharry andNicholas, 1971. Anal. Biochem. 40:1-17); an ³⁵SO₄ ⁻² release assay (seee.g., Seubert, et al., 1985. Arch. Biochem. Biophys. 240: 509-523); anda 32PPi incorporation assay (see e.g., Osslund, et al., 1982. PlantPhysiol. 70: 39-45) have been previously described.

ATP produced by an ATP sulfurylase can be hydrolyzed using enzymaticreactions to generate light. Light-emitting chemical reactions (i.e.,chemiluminescence) and biological reactions (i.e., bioluminescence) arewidely used in analytical biochemistry for sensitive measurements ofvarious metabolites. In bioluminescent reactions, the chemical reactionthat leads to the emission of light is enzyme-catalyzed. For example,the luciferin-luciferase system allows for specific assay of ATP and thebacterial luciferase-oxidoreductase system can be used for monitoring ofNAD(P)H. Both systems have been extended to the analysis of numeroussubstances by means of coupled reactions involving the production orutilization of ATP or NAD(P)H (see e.g., Kricka, 1991. Chemiluminescentand bioluminescent techniques. Clin. Chem. 37: 1472-1281).

The development of new reagents have made it possible to obtain stablelight emission proportional to the concentrations of ATP (see e.g.,Lundin, 1982. Applications of firefly luciferase In; Luminescent Assays(Raven Press, New York) or NAD(P)H (see e.g, Lovgren, et al., Continuousmonitoring of NADH-converting reactions by bacterial luminescence. J.Appl. Biochem. 4: 103-111). With such stable light emission reagents, itis possible to make endpoint assays and to calibrate each individualassay by addition of a known amount of ATP or NAD(P)H. In addition, astable light-emitting system also allows continuous monitoring of ATP-or NAD(P)H-converting systems.

Suitable enzymes for converting ATP into light include luciferases,e.g., insect luciferases. Luciferases produce light as an end-product ofcatalysis. The best known light-emitting enzyme is that of the firefly,Photinus pyralis (Coleoptera). The corresponding gene has been clonedand expressed in bacteria (see e.g., de Wet, et al., 1985. Proc. Natl.Acad. Sci. USA 80: 7870-7873) and plants (see e.g., Ow, et al., 1986.Science 234: 856-859), as well as in insect (see e.g., Jha, et al.,1990. FEBS Lett. 274: 24-26) and mammalian cells (see e.g., de Wet, etal., 1987. Mol. Cell. Biol. 7: 725-7373; Keller, et al., 1987. Proc.Natl. Acad. Sci. USA 82: 3264-3268). In addition, a number of luciferasegenes from the Jamaican click beetle, Pyroplorus plagiophihalamus(Coleoptera), have recently been cloned and partially characterized (seee.g., Wood, et al., 1989. J Biolumin. Chemilumin. 4: 289-301; Wood, etal., 1989. Science 244: 700-702). Distinct luciferases can sometimesproduce light of different wavelengths, which may enable simultaneousmonitoring of light emissions at different wavelengths. Accordingly,these aforementioned characteristics are unique, and add new dimensionswith respect to the utilization of current reporter systems.

Firefly luciferase catalyzes bioluminescence in the presence ofluciferin, adenosine 5′-triphosphate (ATP), magnesium ions, and oxygen,resulting in a quantum yield of 0.88 (see e.g., McElroy and Selinger,1960. Arch. Biochem. Biophys. 88: 136-145). The firefly luciferasebioluminescent reaction can be utilized as an assay for the detection ofATP with a detection limit of approximately 1×10⁻¹³ M (see e.g., Leach,1981. J. Appl. Biochem. 3: 473-517). In addition, the overall degree ofsensitivity and convenience of the luciferase-mediated detection systemshave created considerable interest in the development of fireflyluciferase-based biosensors (see e.g., Green and Kricka, 1984. Talanta31: 173-176; Blum, et al., 1989. J Biolumin. Chemilumin. 4: 543-550).

Using the above-described enzymes, the sequence primer is exposed to apolymerase and a known dNTP. If the dNTP is incorporated onto the 3′ endof the primer sequence, the dNTP is cleaved and a PPi molecule isliberated. The PPi is then converted to ATP with ATP sulfurylase.Preferably, the ATP sulfurylase is present at a sufficiently highconcentration that the conversion of PPi proceeds with first-orderkinetics with respect to PPi. In the presence of luciferase, the ATP ishydrolyzed to generate a photon. The reaction preferably has asufficient concentration of luciferase present within the reactionmixture such that the reaction, ATP→ADP+PO₄ ³⁻+photon (light), proceedswith first-order kinetics with respect to ATP. The photon can bemeasured using methods and apparatuses described below. In oneembodiment, the PPi and a coupled sulfurylase/luciferase reaction isused to generate light for detection. In some embodiments, either orboth the sulfurylase and luciferase are immobilized on one or moremobile solid supports disposed at each reaction site.

The present invention thus permits PPi release to be detected during thepolymerase reaction giving a real-time signal. The sequencing reactionsmay be continuously monitored in real-time. A procedure for rapiddetection of PPi release is thus enabled by the present invention. Thereactions have been estimated to take place in less than 2 seconds(Nyren and Lundin, supra). The rate limiting step is the conversion ofPPi to ATP by ATP sulfurylase, while the luciferase reaction is fast andhas been estimated to take less than 0.2 seconds. Incorporation ratesfor polymerases have also been estimated by various methods and it hasbeen found, for example, that in the case of Klenow polymerase, completeincorporation of one base may take less than 0.5 seconds. Thus, theestimated total time for incorporation of one base and detection by thisenzymatic assay is approximately 3 seconds. It will be seen thereforethat very fast reaction times are possible, enabling real-timedetection. The reaction times could further be decreased by using a morethermostable luciferase.

For most applications it is desirable to use reagents free ofcontaminants like ATP and PPi. These contaminants may be removed byflowing the reagents through a pre-column containing apyrase and/-orpyrophosphatase bound to resin. Alternatively, the apyrase orpyrophosphatase can be bound to magnetic beads and used to removecontaminating ATP and PPi present in the reagents. In addition it isdesirable to wash away diffusible sequencing reagents, e.g.,unincorporated dNTPs, with a wash buffer. Any wash buffer used inpyrophosphate sequencing can be used.

In some embodiments, the concentration of reactants in the sequencingreaction include 1 pmol DNA, 3 pmol polymerase, 40 pmol dNTP in 0.2 mlbuffer. See Ronaghi, et al., Anal. Biochem. 242: 84-89 (1996).

The sequencing reaction can be performed with each of four predeterminednucleotides, if desired. A “complete” cycle generally includessequentially administering sequencing reagents for each of thenucleotides dATP, dGTP, dCTP and dTTP (or dUTP), in a predeterminedorder. Unincorporated dNTPs are washed away between each of thenucleotide additions. Alternatively, unincorporated dNTPs are degradedby apyrase (see below). The cycle is repeated as desired until thedesired amount of sequence of the sequence product is obtained. In someembodiments, about 10-1000, 10-100, 10-75, 20-50, or about 30nucleotides of sequence information is obtained from extension of oneannealed sequencing primer.

In some embodiments, the nucleotide is modified to contain adisulfide-derivative of a hapten such as biotin. The addition of themodified nucleotide to the nascent primer annealed to the anchoredsubstrate is analyzed by a post-polymerization step that includes i)sequentially binding of, in the example where the modification is abiotin, an avidin- or streptavidin-conjugated moiety linked to an enzymemolecule, ii) the washing away of excess avidin- or streptavidin-linkedenzyme, iii) the flow of a suitable enzyme substrate under conditionsamenable to enzyme activity, and iv) the detection of enzyme substratereaction product or products. The hapten is removed in this embodimentthrough the addition of a reducing agent. Such methods enable anucleotide to be identified in a given target position, and the DNA tobe sequenced simply and rapidly while avoiding the need forelectrophoresis and the use of potentially dangerous radiolabels.

A preferred enzyme for detecting the hapten is horse-radish peroxidase.If desired, a wash buffer, can be used between the addition of variousreactants herein. Apyrase can be used to remove unreacted dNTP used toextend the sequencing primer. The wash buffer can optionally includeapyrase.

Example haptens, e.g., biotin, digoxygenin, the fluorescent dyemolecules cy3 and cy5, and fluorescein, are incorporated at variousefficiencies into extended DNA molecules. The attachment of the haptencan occur through linkages via the sugar, the base, and via thephosphate moiety on the nucleotide. Example means for signalamplification include fluorescent, electrochemical and enzymatic. In apreferred embodiment using enzymatic amplification, the enzyme, e.g.alkaline phosphatase (AP), horse-radish peroxidase (HRP),beta-galactosidase, luciferase, can include those for whichlight-generating substrates are known, and the means for detection ofthese light-generating (chemiluminescent) substrates can include a CCDcamera.

In a preferred mode, the modified base is added, detection occurs, andthe hapten-conjugated moiety is removed or inactivated by use of eithera cleaving or inactivating agent. For example, if the cleavable-linkeris a disulfide, then the cleaving agent can be a reducing agent, forexample dithiothreitol (DTT), beta-mercaptoethanol, etc. Otherembodiments of inactivation include heat, cold, chemical denaturants,surfactants, hydrophobic reagents, and suicide inhibitors.

Luciferase can hydrolyze dATP directly with concomitant release of aphoton. This results in a false positive signal because the hydrolysisoccurs independent of incorporation of the dATP into the extendedsequencing primer. To avoid this problem, a dATP analog can be usedwhich is incorporated into DNA, i.e., it is a substrate for a DNApolymerase, but is not a substrate for luciferase. One such analog isα-thio-dATP. Thus, use of α-thio-dATP avoids the spurious photongeneration that can occur when dATP is hydrolyzed without beingincorporated into a growing nucleic acid chain.

Typically, the PPi-based detection is calibrated by the measurement ofthe light released following the addition of control nucleotides to thesequencing reaction mixture immediately after the addition of thesequencing primer. This allows for normalization of the reactionconditions. Incorporation of two or more identical nucleotides insuccession is revealed by a corresponding increase in the amount oflight released. Thus, a two-fold increase in released light relative tocontrol nucleotides reveals the incorporation of two successive dNTPsinto the extended primer.

If desired, apyrase may be “washed” or “flowed” over the surface of thesolid support so as to facilitate the degradation of any remaining,non-incorporated dNTPs within the sequencing reaction mixture. Apyrasealso degrades the generated ATP and hence “turns off” the lightgenerated from the reaction. Upon treatment with apyrase, any remainingreactants are washed away in preparation for the following dNTPincubation and photon detection steps. Alternatively, the apyrase may bebound to the solid or mobile solid support.

Double Ended Sequencing

In a preferred embodiment we provide a method for sequencing from bothends of a nucleic acid template. Traditionally, the sequencing of twoends of a double stranded DNA molecule would require at the very leastthe hybridization of primer, sequencing of one end, hybridization of asecond primer, and sequencing of the other end. The alternative methodis to separate the individual strands of the double stranded nucleicacid and individually sequence each strand. The present inventionprovides a third alternative that is more rapid and less labor intensivethan the first two methods.

The present invention provides for a method of sequential sequencing ofnucleic acids from multiple primers. References to DNA sequencing inthis application are directed to sequencing using a polymerase whereinthe sequence is determined as the nucleotide triphosphate (NTP) isincorporated into the growing chain of a sequencing primer. One exampleof this type of sequencing is the pyro-sequencing detectionpyrophosphate method (see, e.g., U.S. Pat. Nos. 6,274,320, 6,258,568 and6,210,891, each of which is incorporated in total herein by reference.).

In one embodiment, the present invention provides for a method forsequencing two ends of a template double stranded nucleic acid. Thedouble stranded DNA is comprised of two single stranded DNA; referred toherein as a first single stranded DNA and a second single stranded DNA.A first primer is hybridized to the first single stranded DNA and asecond primer is hybridized to the second single stranded DNA. The firstprimer is unprotected while the second primer is protected. “Protection”and “protected” are defined in this disclosure as being the addition ofa chemical group to reactive sites on the primer that prevents a primerfrom polymerization by DNA polymerase. Further, the addition of suchchemical protecting groups should be reversible so that after reversion,the now deprotected primer is once again able to serve as a sequencingprimer. The nucleic acid sequence is determined in one direction (e.g.,from one end of the template) by elongating the first primer with DNApolymerase using conventional methods such as pyrophosphate sequencing.The second primer is then deprotected, and the sequence is determined byelongating the second primer in the other direction (e.g., from theother end of the template) using DNA polymerase and conventional methodssuch as pyrophosphate sequencing. The sequences of the first and secondprimers are specifically designed to hybridize to the two ends of thedouble stranded DNA or at any location along the template in thismethod.

In another embodiment, the present invention provides for a method ofsequencing a nucleic acid from multiple primers. In this method a numberof sequencing primers are hybridized to the template nucleic acid to besequenced. All the sequencing primers are reversibly protected exceptfor one. A protected primer is an oligonucleotide primer that cannot beextended with polymerase and dNTPs which are commonly used in DNAsequencing reactions. A reversibly protected primer is a protectedprimer which can be deprotected. All protected primers referred to inthis invention are reversibly protected. After deprotection, areversibly protected primer functions as a normal sequencing primer andis capable of participating in a normal sequencing reaction.

The present invention provides for a method of sequential sequencing anucleic acid from multiple primers. The method comprises the followingsteps: First, one or more template nucleic acids to be sequenced areprovided. Second, a plurality of sequencing primers are hybridized tothe template nucleic acid or acids. The number of sequencing primers maybe represented by the number n where n can be any positive numbergreater than 1. That number may be, for example, 2, 3, 4, 5, 6, 7, 8, 9,10 or greater. Of the primers, n−1 number may be protected by aprotection group. So, for example, if n is 2, 3, 4, 5, 6, 7, 8, 9 or 10,n−1 would be 1, 2, 3, 4, 5, 6, 7, 8, 9 respectively. The remainingprimer (e.g., n number primers−(n−1) number of protected primers=oneremaining primer) is unprotected. Third, the unprotected primer isextended and the template DNA sequence is determined by conventionalmethods such as, for example, pyrophosphate sequencing. Fourth, afterthe sequencing of the first primer, one of the remaining protectedprimers is unprotected. Fifth, unprotected primer is extended and thetemplate DNA sequence is determined by conventional methods such as, forexample, pyrophosphate sequencing. Optionally, the method may berepeated until sequencing is performed on all the protected primers.

In another aspect, the present invention includes a method of sequentialsequencing of a nucleic acid comprising the steps of: (a) hybridizing 2or more sequencing primers to the nucleic acid wherein all the primersexcept for one are reversibly protected; (b) determining a sequence ofone strand of the nucleic acid by polymerase elongation from theunprotected primer; (c) deprotecting one of the reversibly protectedprimers into an unprotected primer; (d) repeating steps (b) and (c)until all the reversibly protected primers are deprotected and used fordetermining a sequence. In one embodiment, this method comprises oneadditional step between steps (b) and (c), i.e., the step of terminatingthe elongation of the unprotected primer by contacting the unprotectedprimer with DNA polymerase and one or more of a nucleotide triphosphateor a dideoxy nucleotide triphosphate. In yet another embodiment, thismethod further comprises an additional step between said step (b) and(c), i.e., terminating the elongation of the unprotected primer bycontacting the unprotected primer with DNA polymerase and a dideoxynucleotide triphosphate from ddATP, ddTTP, ddCTP, ddGTP or a combinationthereof.

In another aspect, this invention includes a method of sequencing anucleic acid comprising: (a) hybridizing a first unprotected primer to afirst strand of the nucleic acid; (b) hybridizing a second protectedprimer to a second strand; (c) exposing the first and second strands topolymerase, such that the first unprotected primer is extended along thefirst strand; (d) completing the extension of the first sequencingprimer; (e) deprotecting the second sequencing primer; and (f) exposingthe first and second strands to polymerase so that the second sequencingprimer is extended along the second strand. In a preferred embodiment,completing comprises capping or terminating the elongation.

In another embodiment, the present invention provides for a method forsequencing two ends of a template double stranded nucleic acid thatcomprises a first and a second single stranded DNA. In this embodiment,a first primer is hybridized to the first single stranded DNA and asecond primer is hybridized to the second single stranded DNA in thesame step. The first primer is unprotected while the second primer isprotected.

Following hybridization, the nucleic acid sequence is determined in onedirection (e.g., from one end of the template) by elongating the firstprimer with DNA polymerase using conventional methods such aspyrophosphate sequencing. In a preferred embodiment, the polymerase isdevoid of 3′ to 5′ exonuclease activity. The second primer is thendeprotected, and its sequence is determined by elongating the secondprimer in the other direction (e.g., from the other end of the template)with DNA polymerase using conventional methods such as pyrophosphatesequencing. As described earlier, the sequences of the first primer andthe second primer are designed to hybridize to the two ends of thedouble stranded DNA or at any location along the template. Thistechnique is especially useful for the sequencing of many template DNAsthat contain unique sequencing primer hybridization sites on its twoends. For example, many cloning vectors provide unique sequencing primerhybridization sites flanking the insert site to facilitate subsequentsequencing of any cloned sequence (e.g., Bluescript, Stratagene, LaJolla, Calif.).

One benefit of this method of the present invention is that both primersmay be hybridized in a single step. The benefits of this and othermethods are especially useful in parallel sequencing systems wherehybridizations are more involved than normal. Examples of parallelsequencing systems are disclosed in copending U.S. patent applicationSer. No. 10/104,280, the disclosure of which is incorporated in totalherein.

The oligonucleotide primers of the present invention may be synthesizedby conventional technology, e.g., with a commercial oligonucleotidesynthesizer and/or by ligating together subfragments that have been sosynthesized.

In another embodiment of the invention, the length of the doublestranded target nucleic acid may be determined. Methods of determiningthe length of a double stranded nucleic acid are known in the art. Thelength determination may be performed before or after the nucleic acidis sequenced. Known methods of nucleic acid molecule lengthdetermination include gel electrophoresis, pulsed field gelelectrophoresis, mass spectroscopy and the like. Since a blunt endeddouble stranded nucleic acid is comprised of two single strands ofidentical lengths, the determination of the length of one strand of anucleic acid is sufficient to determine the length of the correspondingdouble strand.

The sequence reaction according to the present invention also allows adetermination of the template nucleic acid length. First, a completesequence from one end of the nucleic acid to another end will allow thelength to be determined. Second, the sequence determination of the twoends may overlap in the middle allowing the two sequences to be linked.The complete sequence may be determined and the length may be revealed.For example, if the template is 100 bps long, sequencing from one endmay determine bases 1 to 75; sequencing from the other end may determinebases 25 to 100; there is thus a 51 base overlap in the middle from base25 to base 75; and from this information, the complete sequence from 1to 100 may be determined and the length, of 100 bases, may be revealedby the complete sequence.

Another method of the present invention is directed to a methodcomprising the following steps. First a plurality of sequencing primers,each with a different sequence, is hybridized to a DNA to be sequenced.The number of sequencing primers may be any value greater than one suchas, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. All of theseprimers are reversibly protected except for one. The one unprotectedprimer is elongated in a sequencing reaction and a sequence isdetermined. Usually, when a primer is completely elongated, it cannotextend and will not affect subsequent sequencing from another primer. Ifdesired, the sequenced primer may be terminated using excess polymeraseand dNTP or using ddNTPs. If a termination step is taken, thetermination reagents (dNTPs and ddNTPs) should be removed after thestep. Then, one of the reversibly protected primers is unprotected andsequencing from the second primer proceeds. The steps of deprotecting aprimer, sequencing from the deprotected primer, and optionally,terminating sequencing from the primer is repeated until all theprotected primers are unprotected and used in sequencing.

The reversibly protected primers should be protected with differentchemical groups. By choosing the appropriate method of deprotection, oneprimer may be deprotected without affecting the protection groups of theother primers. In a preferred embodiment, the protection group is PO₄.That is, the second primer is protected by PO₄ and deprotection isaccomplished by T4 polynucleotide kinase (utilizing its 3′-phosphataseactivity). In another preferred embodiment, the protection is a thiogroup or a phosphorothiol group.

The template nucleic acid may be a DNA, RNA, or peptide nucleic acid(PNA). While DNA is the preferred template, RNA and PNA may be convertedto DNA by known techniques such as random primed PCR, reversetranscription, RT-PCR or a combination of these techniques. Further, themethods of the invention are useful for sequencing nucleic acids ofunknown and known sequence. The sequencing of nucleic acid of knownsequence would be useful, for example, for confirming the sequence ofsynthesized DNA or for confirming the identity of suspected pathogenwith a known nucleic acid sequence. The nucleic acids may be a mixtureof more than one population of nucleic acids. It is known that asequencing primer with sufficient specificity (e.g., 20 bases, 25 bases,30 bases, 35 bases, 40 bases, 45 bases, or 50 bases) may be used tosequence a subset of sequences in a long nucleic acid or in a populationof unrelated nucleic acids. Thus, for example, the template may be onesequence of 10 Kb or ten sequences of 1 Kb each. In a preferredembodiment, the template DNA is between 50 bp to 700 bp in length. TheDNA can be single stranded or double stranded.

In the case where the template nucleic acid is single stranded, a numberof primers may be hybridized to the template nucleic acid as shownbelow:

In this case, it is preferred that the initial unprotected primer wouldbe the primer that hybridizes at the most 5′ end of the template. Seeprimer 1 in the above illustration. In this orientation, the elongationof primer 1 would not displace (by strand displacement) primer 2, 3, or4. When sequencing from primer 1 is finished, primer 2 can beunprotected and nucleic acid sequencing can commence. The sequencingfrom primer 2 may displace primer 1 or the elongated version of primerone but would have no effect on the remaining protected primers (primers3 and 4). Using this order, each primer may be used sequentially and asequencing reaction from one primer would not affect the sequencing froma subsequent primer.

One feature of the invention is the ability to use multiple sequencingprimers on one or more nucleic acids and the ability to sequence frommultiple primers using only one hybridization step. In the hybridizationstep, all the sequencing primers (e.g., the n number of sequencingprimers) may be hybridized to the template nucleic acid(s) at the sametime. In conventional sequencing, usually one hybridization step isrequired for sequencing from one primer. One feature of the invention isthat the sequencing from n primers (as defined above) may be performedby a single hybridization step. This effectively eliminates n−1hybridization step.

In a preferred embodiment, the sequences of the n number of primers aresufficiently different that the primers do not cross hybridize orself-hybridize. Cross hybridization refers to the hybridization of oneprimer to another primer because of sequence complementarity. One formof cross hybridization is commonly referred to as a “primer dimer.” Inthe case of a primer dimer, the 3′ ends of two primers are complementaryand form a structure that when elongated, is approximately the sum ofthe length of the two primers. Self-hybridization refers to thesituation where the 5′ end of a primer is complementary to the 3′ end ofthe primer. In that case, the primer has a tendency to self hybridize toform a hairpin-like structure.

A primer can interact or become associated specifically with thetemplate molecule. By the terms “interact” or “associate”, it is meantherein that two substances or compounds (e.g., primer and template;chemical moiety and nucleotide) are bound (e.g., attached, bound,hybridized, joined, annealed, covalently linked, or otherwiseassociated) to one another sufficiently that the intended assay can beconducted. By the terms “specific” or “specifically”, it is meant hereinthat two components bind selectively to each other. The parametersrequired to achieve specific interactions can be determined routinely,e.g., using conventional methods in the art.

To gain more sensitivity or to help in the analysis of complex mixtures,the protected primers can be modified (e.g., derivatized) with chemicalmoieties designed to give clear unique signals. For example, eachprotected primer can be derivatized with a different natural orsynthetic amino acid attached through an amide bond to theoligonucleotide strand at one or more positions along the hybridizingportion of the strand. The chemical modification can be detected, ofcourse, either after having been cleaved from the target nucleic acid,or while in association with the target nucleic acid. By allowing eachprotected target nucleic acid to be identified in a distinguishablemanner, it is possible to assay (e.g., to screen) for a large number ofdifferent target nucleic acids in a single assay. Many such assays canbe performed rapidly and easily. Such an assay or set of assays can beconducted, therefore, with high throughput efficiency as defined herein.

In the methods of the invention, after a first primer is elongated andthe sequence of the template DNA is determined, a second primer isdeprotected and sequenced. There is no interference between thesequencing reaction of the first primer with the sequencing reaction ofthe second, now unprotected, primer because the first primer iscompletely elongated or terminated. Because the first primer iscompletely elongated, the sequencing from the second primer, usingconventional methods such a pyrophosphate sequencing, will not beaffected by the presence of the elongated first primer. The inventionalso provides a method of reducing any possible signal contaminationfrom the first primer. Signal contamination refers to the incidenceswhere the first primer is not completely elongated. In that case, thefirst primer will continue to elongate when a subsequent primer isdeprotected and elongated. The elongation of both the first and secondprimers may interfere with the determination of DNA sequence.

In a preferred embodiment, the sequencing reaction (e.g., the chainelongation reaction) from one primer is first terminated or completedbefore a sequencing reaction is started on a second primer. A chainelongation reaction of DNA can be terminated by contacting the templateDNA with DNA polymerase and dideoxy nucleotide triphosphates (ddNTPs)such as ddATP, ddTTP, ddGTP and ddCTP. Following termination, thedideoxy nucleotide triphosphates may be removed by washing the reactionwith a solution without ddNTPs. A second method of preventing furtherelongation of a primer is to add nucleotide triphosphates (dNTPs such asdATP, dTTP, dGTP and dCTP) and DNA polymerase to a reaction tocompletely extend any primer that is not completely extended. Followingcomplete extension, the dNTPs and the polymerases are removed before thenext primer is deprotected. By completing or terminating one primerbefore deprotecting another primer, the signal to noise ratio of thesequencing reaction (e.g., pyrophosphate sequencing) can be improved.

The steps of (a) optionally terminating or completing the sequencing,(b) deprotecting a new primer, and (c) sequencing from the deprotectedprimer may be repeated until a sequence is determined from theelongation of each primer. In this method, the hybridization stepcomprises “n” number of primers and one unprotected primer. Theunprotected primer is sequenced first and the steps of (a), (b) and (c)above may be repeated.

In a preferred embodiment, pyrophosphate sequencing is used for allsequencing conducted in accordance with the method of the presentinvention.

In another preferred embodiment, the double ended sequencing isperformed according to the process outlined in FIG. 10. This process maybe divided into six steps: (1) creation of a capture bead (FIG. 10A);(2) drive to bead (DTB) PCR amplification (FIG. 10B); (3) SL reportersystem preparation (FIG. 10C); (4) sequencing of the first strand (FIG.10D); (5) preparation of the second strand (FIGS. 10E and 10F); and (6)analysis of each strand (FIG. 10G). This exemplary process is outlinedbelow.

In step 1, an N-hydroxysuccinimide (NHS)-activated capture bead (e.g,Amersham Biosciences, Piscataway, N.J.) is coupled to both a forwardprimer and a reverse primer. NHS coupling forms a chemically stableamide bond with ligands containing primary amino groups. The capturebead is also coupled to biotin (FIG. 10A). The beads (i.e., solidnucleic acid capturing supports) used herein may be of any convenientsize and fabricated from any number of known materials. Example of suchmaterials include: inorganics, natural polymers, and synthetic polymers.Specific examples of these materials include: cellulose, cellulosederivatives, acrylic resins, glass; silica gels, polystyrene, gelatin,polyvinyl pyrrolidone, co-polymers of vinyl and acrylamide, polystyrenecross-linked with divinylbenzene or the like (see, MerrifieldBiochemistry 1964, 3, 1385-1390), polyacrylamides, latex gels,polystyrene, dextran, rubber, silicon, plastics, nitrocellulose,celluloses, natural sponges, silica gels, glass, metals plastic,cellulose, cross-linked dextrans (e.g., Sephadex™) and agarose gel(Sepharose™) and solid phase supports known to those of skill in theart. In a preferred embodiment, the capture beads are Sepharose beadsapproximately 25 to 40 μM in diameter.

In step 2, template DNA which has hybridized to the forward and reverseprimers is added, and the DNA is amplified through a PCR amplificationstrategy (FIG. 10B). In one embodiment, the DNA is amplified by EmulsionPolymerase Chain Reaction, Drive to Bead Polymerase Chain Reaction,Rolling Circle Amplification or Loop-mediated Isothermal Amplification.In step 3, streptavidin is added followed by the addition of sulfurylaseand luciferase which are coupled to the streptavidin (FIG. 10C). Theaddition of auxiliary enzymes during a sequencing method has beendisclosed in U.S. Ser. No. 10/104,280 and U.S. Ser. No. 10/127,906,which are incorporated herein in their entireties by reference. In oneembodiment, the template DNA has a DNA adaptor ligated to both the 5′and 3′ end. In a preferred embodiment, the DNA is coupled to the DNAcapture bead by hybridization of one of the DNA adaptors to acomplimentary sequence on the DNA capture bead.

In the first step, single stranded nucleic acid template to be amplifiedis attached to a capture bead. The nucleic acid template may be attachedto the capture bead in any manner known in the art. Numerous methodsexist in the art for attaching the DNA to a microscopic bead. Covalentchemical attachment of the DNA to the bead can be accomplished by usingstandard coupling agents, such as water-soluble carbodiimide, to linkthe 5′-phosphate on the DNA to amine-coated microspheres through aphosphoamidate bond. Another alternative is to first couple specificoligonucleotide linkers to the bead using similar chemistry, and to thenuse DNA ligase to link the DNA to the linker on the bead. Other linkagechemistries include the use of N-hydroxysuccinamide (NHS) and itsderivatives, to join the oligonucleotide to the beads. In such a method,one end of the oligonucleotide may contain a reactive group (such as anamide group) which forms a covalent bond with the solid support, whilethe other end of the linker contains another reactive group which canbond with the oligonucleotide to be immobilized. In a preferredembodiment, the oligonucleotide is bound to the DNA capture bead bycovalent linkage. However, non-covalent linkages, such as chelation orantigen-antibody complexes, may be used to join the oligonucleotide tothe bead.

Oligonucleotide linkers can be employed which specifically hybridize tounique sequences at the end of the DNA fragment, such as the overlappingend from a restriction enzyme site or the “sticky ends” of bacteriophagelambda based cloning vectors, but blunt-end ligations can also be usedbeneficially. These methods are described in detail in U.S. Pat. No.5,674,743, the disclosure of which is incorporated in toto herein. It ispreferred that any method used to immobilize the beads will continue tobind the immobilized oligonucleotide throughout the steps in the methodsof the invention. In a preferred embodiment, the oligonucleotide isbound to the DNA capture bead by covalent linkage. However, non-covalentlinkages, such as chelation or antigen-antibody complexes, may be usedto join the oligonucleotide to the bead.

In step 4, the first strand of DNA is sequenced by depositing thecapture beads onto a PicoTiter plate (PTP), and sequencing by a methodknown to one of ordinary skill in the art (e.g., pyrophosphatesequencing) (FIG. 10D). Following sequencing, a mixture of dNTPs andddNTPs are added in order to “cap” or terminate the sequencing process(FIG. 10E). In step 5, the second strand of nucleic acid is prepared byadding apyrase to remove the ddNTPs and polynucleotide kinase (PNK) toremove the 3′ phosphate group from the blocked primer strand (FIG. 10F).Polymerase is then added to prime the second strand followed bysequencing of the second strand according to a standard method known toone of ordinary skill in the art (FIG. 10G). In step 7, the sequence ofthe both the first and second strand is analyzed such that a contiguousDNA sequence is determined.

Detection Means

The solid support is optically linked to an imaging system 230, whichincludes a CCD system in association with conventional optics or a fiberoptic bundle. In one embodiment the perfusion chamber substrate includesa fiber optic array wafer such that light generated near the aqueousinterface is transmitted directly through the optical fibers to theexterior of the substrate or chamber. When the CCD system includes afiber optic connector, imaging can be accomplished by placing theperfusion chamber substrate in direct contact with the connector.Alternatively, conventional optics can be used to image the light, e.g.,by using a 1-1 magnification high numerical aperture lens system, fromthe exterior of the fiber optic substrate directly onto the CCD sensor.When the substrate does not provide for fiber optic coupling, a lenssystem can also be used as described above, in which case either thesubstrate or the perfusion chamber cover is optically transparent. Anexemplary CCD imaging system is described above.

The imaging system 230 is used to collect light from the reactors on thesubstrate surface. Light can be imaged, for example, onto a CCD using ahigh sensitivity low noise apparatus known in the art. For fiber-opticbased imaging, it is preferable to incorporate the optical fibersdirectly into the cover slip or for a FORA to have the optical fibersthat form the microwells also be the optical fibers that convey light tothe detector.

The imaging system is linked to a computer control and data collectionsystem 240. In general, any commonly available hardware and softwarepackage can be used. The computer control and data collection system isalso linked to the conduit 200 to control reagent delivery.

The photons generated by the pyrophosphate sequencing reaction arecaptured by the CCD only if they pass through a focusing device (e.g.,an optical lens or optical fiber) and are focused upon a CCD element.However, the emitted photons will escape equally in all directions. Inorder to maximize their subsequent “capture” and quantitation whenutilizing a planar array (e.g., a DNA chip), it is preferable to collectthe photons as close as possible to the point at which they aregenerated, e.g. immediately at the planar solid support. This isaccomplished by either: (i) utilizing optical immersion oil between thecover slip and a traditional optical lens or optical fiber bundle or,preferably, (ii) incorporating optical fibers directly into the coverslip itself. Similarly, when a thin, optically transparent planarsurface is used, the optical fiber bundle can also be placed against itsback surface, eliminating the need to “image” through the depth of theentire reaction/perfusion chamber.

The reaction event, e.g., photons generated by luciferase, may bedetected and quantified using a variety of detection apparatuses, e.g.,a photomultiplier tube, a CCD, CMOS, absorbance photometer, aluminometer, charge injection device (CID), or other solid statedetector, as well as the apparatuses described herein. In a preferredembodiment, the quantitation of the emitted photons is accomplished bythe use of a CCD camera fitted with a fused fiber optic bundle. Inanother preferred embodiment, the quantitation of the emitted photons isaccomplished by the use of a CCD camera fitted with a microchannel plateintensifier. A back-thinned CCD can be used to increase sensitivity. CCDdetectors are described in, e.g., Bronks, et al., 1995. Anal. Chem. 65:2750-2757.

An exemplary CCD system is a Spectral Instruments, Inc. (Tucson, Ariz.)Series 600 4-port camera with a Lockheed-Martin LM485 CCD chip and a 1-1fiber optic connector (bundle) with 6-8 μm individual fiber diameters.This system has 4096×4096, or greater than 16 million pixels and has aquantum efficiency ranging from 10% to >40%. Thus, depending onwavelength, as much as 40% of the photons imaged onto the CCD sensor areconverted to detectable electrons.

In other embodiments, a fluorescent moiety can be used as a label andthe detection of a reaction event can be carried out using a confocalscanning microscope to scan the surface of an array with a laser orother techniques such as scanning near-field optical microscopy (SNOM)are available which are capable of smaller optical resolution, therebyallowing the use of “more dense” arrays. For example, using SNOM,individual polynucleotides may be distinguished when separated by adistance of less than 100 nm, e.g., 10 nm×10 nm. Additionally, scanningtunneling microscopy (Binning et al., Helvetica Physica Acta,55:726-735, 1982) and atomic force microscopy (Hanswa et al., Annu RevBiophys Biomol Struct, 23:115-139, 1994) can be used.

Haplotype Application

Virtually any sequencing application can be accomplished using themethods and apparatus of this invention. In one embodiment wecontemplate haplotype mapping. Human gene diversity is an importantfactor in the variability of patient response to pharmaceuticals. Themost precise measurement of this diversity is the haplotype, which isthe organization of polymorphic variation as it is found on achromosome. Recently, major government and academic genome researchersin the US, Canada and Europe have agreed that haplotypes are a powerfultool that can reduce the complexity of genetic information to apractical form. Haplotypes can be used in drug discovery to improve theoutcome of target validation and drug screening studies and in drugdevelopment to improve the design and reliability of clinical trials.Haplotype markers can be used to predict the efficacy and safety of newand approved drugs and will serve as the foundation for a new paradigmof personalized medicine matching patients to the right drug at theright dose via guidance from a database of clinical marker-associations.

Numerous empirical studies have shown that nearby SNP alleles are oftenin linkage disequilibrium (LD) with each other, such that the state ofone SNP allele is often highly correlated with the allele of anotherclose SNP. These correlations exist because of the shared history oftightly linked SNP'S, which are co-transmitted from generation togeneration. Patterns of human sequence variation (haplotypes) thusrepresent ancestral DNA segments. Historical meioses have slowlydissociated alleles from neighboring alleles on ancestral chromosomes,except for tightly linked variants. The extent of linkage disequilibriumin founder populations with recent bottlenecks had been the object ofnumerous studies—particularly in the cloning of simple Mendeliandisorders such as cystic fibrosis (16), Huntington's disease (11),diastrophic dysplasia (DTD) (8). Whereas these cloning studies benefitedfrom the large chromosomal segments showing LD spanning over largedistances (often in the megabase range), very little empirical data wasavailable until recently regarding LD across the human genome in theworld population.

We focus on three recent examples of large-scale surveys of LD (andhaplotypes): (see, e.g, Reich, D. E., Cargill, M., Bolk, S., Ireland,J., Sabeti, P. C., Richter, D. J., Layery, T., Kouyoumjian, R.,Farhadian, S. F., Ward, R. & Lander, E. S. 2001. Linkage disequilibriumin the human genome. Nature 411, 199-204.26). We sampled 19 chromosomeregions for their SNP content. High frequency SNP's spanning intervalsof 2 to 160 kb were first genotyped in a Caucasian samples. Over allregions, LD was detectable at distances of about 60 kb, with asignificant difference between regions, as the range was as short as 6kb at one locus and as long 155 kb in another. Not surprisingly, LD wassignificantly correlated with the estimated local recombination rates.Further analysis in a Nigerian sample provided evidence of shorter LD inthis population—although the allelic combinations over short distanceswere similar to the Caucasian sample. Overall—this work providedevidence that large blocks of LD are common across the human genome, andthat genome-wide LD mapping of disease genes will be feasible.

Kits

The invention also comprises kits for use in methods of the inventionwhich could include one or more of the following components: (a) a testspecific primer which hybridizes to sample DNA so that the targetposition is directly adjacent to the 3′ end of the primer; (b) apolymerase; (c) detection enzyme means for identifying PPi release; (d)deoxynucleotides including, in place of dATP, a dATP analogue which iscapable of acting as a substrate for a polymerase but incapable ofacting as a substrate for a said PPi-detection enzyme; and (e)optionally dideoxynucleotides, optionally ddATP being replaced by addATP analogue which is capable of acting as a substrate for apolymerase but incapable of acting as a substrate for a saidPPi-detection enzyme. If the kit is for use with initial PCRamplification then it could also include the following components: (i) apair of primers for PCR, at least one primer having means permittingimmobilization of said primer; (ii) a polymerase which is preferablyheat stable, for example Taq I polymerase; (iii) buffers for the PCRreaction; and (iv) deoxynucleotides. Where an enzyme label is used toevaluate PCR, the kit will advantageously contain a substrate for theenzyme and other components of a detection system.

One embodiment of the invention is directed to a method for sequencingnucleic acids. The method involves fragmenting large template nucleicacid molecules to generate a plurality of fragmented nucleic acids. Thenthe fragmented nucleic acids are delivered into aqueous microreactors ina water-in-oil emulsion such that a plurality of aqueous microreactorscomprise a single copy of a fragmented nucleic acid, a single beadcapable of binding to the fragmented nucleic acid, and amplificationreaction solution containing reagents necessary to perform nucleic acidamplification. In the next step, the fragmented nucleic acids isamplified in the microreactors to form amplified copies of the nucleicacids and binding the amplified copies to beads in the microreactors.Next, the beads are delivered to an array of at least 10,000 reactionchambers on a planar surface, wherein a plurality of the reactionchambers comprise no more than a single bead. Finally, a sequencingreaction is performed simultaneously on a plurality of the reactionchambers.

Another embodiment of the invention is directed to an array comprising aplanar surface with a plurality of cavities thereon, each cavity formingan analyte reaction chamber, wherein the reaction chambers have a centerto center spacing of between 20 to 100 μm and each cavity has a width inat least one dimension of between 20 μm and 70 μm. Further, there are atleast 10,000 reaction chambers in the array. Each reaction chambers maycontain at least 100,000 copies of a single species of single strandednucleic acid template.

Another embodiment of the invention is directed to an array comprising aplanar top surface and a planar bottom surface wherein the planar topsurface has at least 10,000 cavities thereon, each cavity forming ananalyte reaction chamber, the planar bottom surface is opticallyconductive such that optical signals from the reaction chambers can bedetected through the bottom planar surface, wherein the distance betweenthe top surface and the bottom surface is no greater than 5 mm, whereinthe reaction chambers have a center to center spacing of between 20 to100 μm and each chamber having a width in at least one dimension ofbetween 20 μm and 70 μm. The distance between the top surface and thebottom surface, in one embodiment, is no greater than 2 mm.

Another embodiment of the invention is directed to an array means forcarrying out separate parallel common reactions in an aqueousenvironment. The array means may comprise a substrate comprising atleast 10,000 discrete reaction chambers containing a starting materialthat is capable of reacting with a reagent, each of the reactionchambers being dimensioned such that when one or more fluids containingat least one reagent is delivered into each reaction chamber, thediffusion time for the reagent to diffuse out of the well exceeds thetime required for the starting material to react with the reagent toform a product.

Another embodiment of the invention is directed to a method fordelivering a bioactive agent to an array. The method comprisesdispersing over the array a plurality of mobile solid supports, eachmobile solid support having at least one reagent immobilized thereon,wherein the reagent is suitable for use in a nucleic acid sequencingreaction, where the array comprises a planar surface with a plurality ofreaction chambers disposed thereon. The reaction chambers may have acenter to center spacing of between 20 to 100 μm and each reactionchamber has a width in at least one dimension of between 20 μm and 70μm.

Another embodiment of the invention is directed to an apparatus forsimultaneously monitoring an array of reaction chambers for lightindicating that a reaction is taking place at a particular site. Theapparatus comprises (a) an array of reaction chambers formed from aplanar substrate comprising a plurality of cavitated surfaces, eachcavitated surface forming a reaction chamber adapted to containanalytes, and wherein the reaction chambers have a center to centerspacing of between 20 to 100 μm, each reaction chamber having a volumeof between 10 to 150 pL, the array comprising more than 10,000 discretereaction chambers; (b) an optically sensitive device arranged so that inuse the light from a particular reaction chamber will impinge upon aparticular predetermined region of the optically sensitive device; (c)means for determining the light level impinging upon each of thepredetermined regions; and (d) means to record the variation of thelight level with time for each of the reaction chamber.

Another embodiment of the invention is directed to an analytic sensor,comprising (a) an array formed from a first bundle of optical fiberswith a plurality of cavitated surfaces at one end thereof, eachcavitated surface forming a reaction chamber adapted to containanalytes, and wherein the reaction chambers have a center to centerspacing of between 20 to 100 μm, a width of 20 to 70 μm, the arraycomprising more than 10,000 discrete reaction chambers; (b) an enzymaticor fluorescent means for generating light in the reaction chambers; and(c) light detection means comprising a light capture means and a secondfiber optic bundle for transmitting light to the light detecting means,the second fiber optic bundle being in optical contact with the array,such that light generated in an individual reaction chamber is capturedby a separate fiber or groups of separate fibers of the second fiberoptic bundle for transmission to the light capture means.

Another embodiment of the invention is directed to a method for carryingout separate parallel common reactions in an aqueous environment. Thefirst step involves delivering a fluid containing at least one reagentto an array, wherein the array comprises a substrate comprising at least10,000 discrete reaction chambers, each reaction chamber adapted tocontain analytes, and wherein the reaction chambers have a volume ofbetween 10 to 150 pL and containing a starting material that is capableof reacting with the reagent, each of the reaction chambers beingdimensioned such that when the fluid is delivered into each reactionchamber, the diffusion time for the reagent to diffuse out of the wellexceeds the time required for the starting material to react with thereagent to form a product. The second step involves washing the fluidfrom the array in the time period (i) after the starting material hasreacted with the reagent to form a product in each reaction chamber but(ii) before the reagent delivered to any one reaction chamber hasdiffused out of that reaction chamber into any other reaction chamber.

Another embodiment of the invention is directed to a method fordelivering nucleic acid sequencing enzymes to an array. The array havinga planar surface with a plurality of cavities thereon, each cavityforming an analyte reaction chamber, wherein the reaction chambers havea center to center spacing of between 20 to 100 μm. The method involvesthe step of dispersing over the array a plurality of mobile solidsupports having one or more nucleic acid sequencing enzymes immobilizedthereon, such that a plurality of the reaction chambers contain at leastone mobile solid support.

Another embodiment of the invention is directed to a method fordelivering a plurality of nucleic acid templates to an array. The arraymay have a planar surface with a plurality of cavities thereon, eachcavity forming an analyte reaction chamber, wherein the reactionchambers have a center to center spacing of between 20 to 100 μm and thearray having at least 10,000 reaction chambers. The method comprise thestep of dispersing over the array a plurality of mobile solid supports,each mobile solid support having no more than a single species ofnucleic acid template immobilized thereon, the dispersion causing nomore than one mobile solid support to be disposed within any onereaction chamber.

Another embodiment of the invention is directed to a method forsequencing a nucleic acid. The method comprises the step of providing aplurality of single-stranded nucleic acid templates disposed within aplurality of cavities on a planar surface, each cavity forming ananalyte reaction chamber, wherein the reaction chambers have a center tocenter spacing of between 20 to 100 μm and the planar surface has atleast 10,000 reaction chambers. The next step involves performing apyrophosphate based sequencing reaction simultaneously on all reactionchambers by annealing an effective amount of a sequencing primer to thenucleic acid templates and extending the sequencing primer with apolymerase and a predetermined nucleotide triphosphate to yield asequencing product and, if the predetermined nucleotide triphosphate isincorporated onto the 3′ end of the sequencing primer, a sequencingreaction byproduct. The third step involves identifying the sequencingreaction byproduct, thereby determining the sequence of the nucleic acidin each reaction chamber.

Another embodiment of the invention is directed to a method ofdetermining the base sequence of a plurality of nucleotides on an array.The first step involves providing at least 10,000 DNA templates, eachseparately disposed within a plurality of cavities on a planar surface,each cavity forming an analyte reaction chamber, wherein the reactionchambers have a center to center spacing of between 20 to 100 μm, and avolume of between 10 to 150 pL. The second step involves adding anactivated nucleotide 5′-triphosphate precursor of one known nitrogenousbase to a reaction mixture in each reaction chamber, each reactionmixture comprising a template-directed nucleotide polymerase and asingle-stranded polynucleotide template hybridized to a complementaryoligonucleotide primer strand at least one nucleotide residue shorterthan the templates to form at least one unpaired nucleotide residue ineach template at the 3′-end of the primer strand, under reactionconditions which allow incorporation of the activated nucleoside5′-triphosphate precursor onto the 3′-end of the primer strands,provided the nitrogenous base of the activated nucleoside5′-triphosphate precursor is complementary to the nitrogenous base ofthe unpaired nucleotide residue of the templates. The third stepinvolves detecting whether or not the nucleoside 5′-triphosphateprecursor was incorporated into the primer strands in whichincorporation of the nucleoside 5′-triphosphate precursor indicates thatthe unpaired nucleotide residue of the template has a nitrogenous basecomposition that is complementary to that of the incorporated nucleoside5′-triphosphate precursor. The fourth step involves sequentiallyrepeating steps (b) and (c), wherein each sequential repetition addsand, detects the incorporation of one type of activated nucleoside5′-triphosphate precursor of known nitrogenous base composition. Thefifth step involves determining the base sequence of the unpairednucleotide residues of the template in each reaction chamber from thesequence of incorporation of the nucleoside precursors.

Another embodiment of the invention is directed to a method ofidentifying the base in a target position in a DNA sequence of templateDNA. The first step involves providing at least 10,000 separate DNAtemplates are separately disposed within a plurality of cavities on aplanar surface, each cavity forming an analyte reaction chamber, whereinthe reaction chambers have a center to center spacing of between 20 to100 μm, the DNA being rendered single stranded either before or afterbeing disposed in the reaction chambers. The second step involvesproviding an extension primer is provided which hybridizes to theimmobilized single-stranded DNA at a position immediately adjacent tothe target position. The immobilized single-stranded DNA is subjected toa polymerase reaction in the presence of a predetermined deoxynucleotideor dideoxynucleotide, wherein if the predetermined deoxynucleotide ordideoxynucleotide is incorporated onto the 3′ end of the sequencingprimer then a sequencing reaction byproduct is formed. The fourth stepinvolves identifying the sequencing reaction byproduct, therebydetermining the nucleotide complementary to the base at the targetposition in each of the 10,000 DNA templates.

Another embodiment of the invention is directed to an apparatus foranalyzing a nucleic acid sequence. The apparatus comprises: (a) areagent delivery cuvette, wherein the cuvette includes an arraycomprising a planar surface with a plurality of cavities thereon, eachcavity forming an analyte reaction chamber, wherein the reactionchambers have a center to center spacing of between 20 to 100 μm, andthere are in excess of 10,000 reaction chambers, and wherein the reagentdelivery cuvette contains reagents for use in a sequencing reaction; (b)a reagent delivery means in communication with the reagent deliverycuvette; (c) an imaging system in communication with the reagentdelivery chamber; and (d) a data collection system in communication withthe imaging system.

Another embodiment of the invention is directed to an apparatus fordetermining the base sequence of a plurality of nucleotides on an array.The apparatus comprises: (a) a reagent cuvette containing a plurality ofcavities on a planar surface, each cavity forming an analyte reactionchamber, wherein there are in excess of 10,000 reaction chambers, eachhaving a center to center spacing of between 20 to 100 μm and a volumeof between 10 to 150 pL; (b) reagent delivery means for simultaneouslyadding to each reaction chamber an activated nucleotide 5′-triphosphateprecursor of one known nitrogenous base to a reaction mixture in eachreaction chamber, each reaction mixture comprising a template-directednucleotide polymerase and a single-stranded polynucleotide templatehybridized to a complementary oligonucleotide primer strand at least onenucleotide residue shorter than the templates to form at least oneunpaired nucleotide residue in each template at the 3′-end of the primerstrand, under reaction conditions which allow incorporation of theactivated nucleoside 5′-triphosphate precursor onto the 3′-end of theprimer strands, provided the nitrogenous base of the activatednucleoside 5′-triphosphate precursor is complementary to the nitrogenousbase of the unpaired nucleotide residue of the templates; (c) detectionmeans for detecting in each reaction chamber whether or not thenucleoside 5′-triphosphate precursor was incorporated into the primerstrands in which incorporation of the nucleoside 5′-triphosphateprecursor indicates that the unpaired nucleotide residue of the templatehas a nitrogenous base composition that is complementary to that of theincorporated nucleoside 5′-triphosphate precursor; and (d) means forsequentially repeating (b) and (c), wherein each sequential repetitionadds and, detects the incorporation of one type of activated nucleoside5′-triphosphate precursor of known nitrogenous base composition; and (e)data processing means for determining the base sequence of the unpairednucleotide residues of the template simultaneously in each reactionchamber from the sequence of incorporation of the nucleoside precursors.

Another embodiment of the invention is directed to an apparatus forprocessing a plurality of analytes. The apparatus comprises: (a) a flowchamber having disposed therein a substrate comprising at least 50,000cavitated surfaces on a fiber optic bundle, each cavitated surfaceforming a reaction chamber adapted to contain analytes, and wherein thereaction chambers have a center to center spacing of between 20 to 100μm and a diameter of 20 to 70 μm; (b) fluid means for deliveringprocessing reagents from one or more reservoirs to the flow chamber sothat the analytes disposed in the reaction chambers are exposed to thereagents; and (c) detection means for simultaneously detecting asequence of optical signals from each of the reaction chambers, eachoptical signal of the sequence being indicative of an interactionbetween a processing reagent and the analyte disposed in the reactionchamber, wherein the detection means is in communication with thecavitated surfaces.

Another embodiment of the invention is directed to a method forsequencing a nucleic acid. The first step involves providing a pluralityof single-stranded nucleic acid templates in an array having at least50,000 discrete reaction sites. The second step involves contacting thenucleic acid templates with reagents necessary to perform apyrophosphate-based sequencing reaction coupled to light emission. Thethird step involves detecting the light emitted from a plurality ofreaction sites on respective portions of an optically sensitive device.The forth step involves converting the light impinging upon each of theportions of the optically sensitive device into an electrical signalwhich is distinguishable from the signals from all of the other reactionsites. The fifth step involves determining the sequence of the nucleicacid templates based on light emission for each of the discrete reactionsites from the corresponding electrical signal.

Another embodiment of the invention is directed to a method forsequencing nucleic acids. The first step involves fragmenting largetemplate nucleic acid molecules to generate a plurality of fragmentednucleic acids. The second step involves attaching one strand of aplurality of the fragmented nucleic acids individually to beads togenerate single stranded nucleic acids attached individually to beads.The third step involves delivering a population of the single strandedfragmented nucleic acids attached individually to beads to an array ofat least 10,000 reaction chambers on a planar surface, wherein aplurality of the wells comprise no more than a one bead with on singlestranded fragmented nucleic acid. The fourth step involves performing asequencing reaction simultaneously on a plurality of the reactionchambers. The sequencing reaction may have the steps of (a) annealing aneffective amount of a sequencing primer to the single strandedfragmented nucleic acid templates and extending the sequencing primerwith a polymerase and a predetermined nucleotide triphosphate to yield asequencing product and, if the predetermined nucleotide triphosphate isincorporated onto the 3′ end of the sequencing primer, a sequencingreaction byproduct; and (b) identifying the sequencing reactionbyproduct, thereby determining the sequence of the nucleic acid in aplurality of the reaction chambers. Alternatively, the sequencingreaction may comprises the steps of: (a) hybridizing two or moresequencing primers to one or a plurality of single strands of thenucleic acid molecule wherein all the primers except for one arereversibly blocked primers; (b) incorporating at least one base onto thenucleic acid molecule by polymerase elongation from an unblocked primer;(c) preventing further elongation of the unblocked primer; (d)deblocking one of the reversibly blocked primers into an unblockedprimer; and (e) repeating steps (b) to (d) until at least one of thereversibly blocked primers are deblocked and used for determining asequence.

Other materials and methods may be found in the following copending USpatent applications: U.S. Ser. No. 60/443,471 filed Jan. 29, 2003 andU.S. Ser. No. 60/465,071 filed Apr. 23, 2003. All patents, patentapplications, and references cited in this disclosure are incorporatedherein by reference.

EXAMPLES Example 1 Sample Preparation

DNA Sample:

The DNA should be of high quality and free from contaminants such asproteins, nucleases, lipids, and other chemicals (such as residual EDTAfrom preparation) and salts. It is preferred that genomic DNA shouldhave a 260/280 ratio of 1.8 or higher. If it is desired to sequence thegenome of only one organism, then the DNA should be quality checked toensure that there is no contaminating DNA. For example: a preparation ofhuman DNA may be checked by PCR to ensure that it is not contaminated bybacterial DNA molecules. Another method of checking for contamination isby restriction digestion patterns and especially restriction digestionfollowed by Southern Blot using suitable probes known to be specific foran organism (e.g., human or mouse) and a second probe known to bespecific for a possible contaminating organism (e.g., E. coli). If it isdesired, the DNA should originate from a single clone of the organism(e.g., a colony if from bacteria).

Step 1: DNase I Digestion

The purpose of the DNase I digestion step is to fragment a large stretchof DNA such as a whole genome or a large portion of a genome intosmaller species. This population of smaller-sized DNA species generatedfrom a single DNA template is referred to as a “library”.Deoxyribonuclease I (DNase I) is an endonuclease which cleavesdouble-stranded template DNA. The cleavage characteristics of DNase Iallow random digestion of template DNA (i.e., minimal sequence bias) andwill result in the predominance of blunt-ended, double-stranded DNAfragments when used in the presence of manganese-based buffers (Melgarand Goldthwait 1968). The digestion of genomic templates by DNase I isdependent on three factors: i) quantity of enzyme used (units); ii)temperature of digestion (° C.); and iii) incubation time (minutes). TheDNase I digestion conditions outlined below were optimized to yield DNAlibraries in a size range from 50-700 base pairs (bp).

1. DNA was obtained and prepared to a concentration of 0.3 mg/ml inTris-HCl (10 mM, pH 7-8). A total of 134 μl of DNA (15 μg) was neededfor this preparation. It is recommended to not use DNA preparationsdiluted with buffers containing EDTA (i.e., TE, Tris/EDTA). The presenceof EDTA is inhibitory to enzyme digestion with DNase I. If the DNApreparation contains EDTA, it is important that the DNA be “salted” outof solution and reconstituted with the appropriate Tris-HCl buffer (10mM, pH 7-8) or nanopure H₂O (pH 7-8).

2. In a 0.2 ml tube, DNase I Buffer, comprising 50 μl Tris pH 7.5 (1M),10 μl MnCl₂ (1M), 1 μl BSA (100 mg/ml), and 39 μl water was prepared.

3. In a separate 0.2 ml tube, 15 μl of DNase I Buffer and 1.5 μl ofDNase I (1 U/ml) was added. The reaction tube was placed in a thermalcycler set to 15° C.

4. The 134 μl of DNA (0.3 mg/ml) was added to the DNase I reaction tubeplaced in the thermal cycler set at 15° C. The lid was closed and thesample was incubated for exactly 1 minute. Following incubation, 50 μlof 50 mM EDTA was added to stop the enzyme digestion.

5. The digested DNA was purified by using the QiaQuick PCR purificationkit. The digestion reaction was then split into four aliquots, and fourspin columns were used to purify each aliquot (37.5 μl per spin column).Each column was eluted with 30 μl elution buffer (EB) according to themanufacturer's protocol. The eluates were then combined to generate afinal reaction volume of 120 μl.

6. One 3 μl aliquot of the digestion reaction was saved for analysisusing a BioAnalzyer DNA 1000 LabChip.

Step 2: Pfu Polishing

Digestion of DNA templates with DNase I yields fragments of DNA that areprimarily blunt-ended, however, some fragments will have ends thatcontain protruding termini that are one or two nucleotides in length.Pfu polishing is used to increase the amount of blunt-ended species byfill-in (i.e., “blunting”) of 5′ overhangs. Additionally, Pfu DNApolymerase has 3′→5′ exonuclease activity that will result in theremoval of single and double nucleotide extensions. Pfu polishingincreases the amount of blunt-ended DNA fragments available for adaptorligation (Costa 1994a, 1994b, 1994c). The following Pfu polishingprotocol was used.

1. In a 0.2 ml tube, 115 μl purified, DNase I-digested DNA fragments, 15μl 10× Cloned Pfu buffer, 5 μl dNTPs (10 mM), and 15 μl cloned Pfu DNApolymerase (2.5 U/μl) were added in order.

2. The polishing reaction components were mixed well and incubated at72° C. for 30 minutes.

3. Following incubation, the reaction tube was removed and placed on icefor 2 minutes.

4. The polishing reaction mixture was then split into four aliquots andpurified using QiaQuick PCR purification columns (37.5 μL on eachcolumn). Each column was eluted with 30 μl buffer EB according to themanufacturer's protocol. The eluates were then combined to generate afinal reaction volume of 120 μL.

5. One 3 μl aliquot of the final polishing reaction was saved foranalysis using a BioAnalzyer DNA 1000 LabChip.

Step 3: Ligation of Universal Adaptors to Fragmented DNA Library

Following fragmentation and polishing of the genomic DNA library, primersequences are added to the ends of each DNA fragment. These primersequences are termed “Universal Adaptors” and are comprised ofdouble-stranded oligonucleotides that contain specific priming regionsthat afford both PCR amplification and nucleotide sequencing. TheUniversal Adaptors are designed to include a set of unique PCR primingregions that are 20 base pairs in length located adjacent to a set ofunique sequencing priming regions that are 20 base pairs in length,followed by a unique 4-base “key” consisting of one of eachdeoxyribonucleotide (i.e., A, C, G, T). Each unique Universal Adaptor(termed “Universal Adaptor A” and “Universal Adaptor B”) is forty-fourbase pairs (44 bp) in length. Universal Adaptors are ligated, using T4DNA ligase, onto each end of the DNA fragment to generate a totalnucleotide addition of 88 bp to each DNA fragment. Different UniversalAdaptors are designed specifically for each genomic DNA librarypreparation and will therefore provide a unique identifier for eachorganism.

To prepare a pair of Universal Adaptors, single-strandedoligonucleotides are designed in-house and are manufactured through acommercial vendor. Universal Adaptor DNA oligonucleotides are designedwith two phosphorothioate linkages at each oligonucleotide end thatserve to protect against nuclease activity (Samini, T. D., B. Jolles,and A. Laigle. 2001. Best minimally modified antisense oligonucleotidesaccording to cell nuclease activity. Antisense Nucleic Acid Drug Dev.11(3):129., the disclosure of which is incorporated in toto herein byreference.). Each oligonucleotide is HPLC-purified to ensure there areno contaminating or spurious DNA oligonucleotide sequences in the finalprep.

The Universal Adaptors are designed to allow directional ligation to theblunt-ended, fragmented genomic DNA. For each Universal Adaptor pair,the PCR priming region contains a 5′ four-base overhang and ablunt-ended 3′ Key region. Directionality is achieved as the blunt-endside of the Universal Adaptor ligates to the blunt-ended DNA fragmentwhile the 5′ overhang of the adaptor cannot ligate to the blunt-endedDNA fragment. Additionally, a 5′ biotin is added to the UniversalAdaptor B to allow subsequent isolation of ssDNA template (Step 8). EachUniversal Adaptor is prepared by annealing, in a single tube, the twosingle-stranded complementary DNA oligonucleotides (i.e., one oligocontaining the sense sequence and the second oligo containing theantisense sequence). The following ligation protocol was used.

1. In a 0.2 ml tube, 39 μl nH₂O (molecular biology grade water), 25 μldigested, polished DNA Library, 100 μl 2× Quick Ligase Reaction Buffer,20 μl MMP1 (10 pm/μl) adaptor set, 100:1 ratio, and 16 μl Quick Ligasewere added in order. The ligation reaction was mixed well and incubatedat RT for 20 minutes.

2. The ligation reaction was then removed and a 10-μl aliquot of theligation reaction was purified for use on the BioAnalyzer. A single spincolumn from the Qiagen Min-Elute kit was used. The column was elutedwith 10 μl EB according to the procedure per manufacturers' protocol. A1-μl aliquot of the purified ligation reaction was loaded using aBioAnalyzer DNA 1000 LabChip. This purification step is recommended asthe unpurified ligation reaction contains high amounts of salt and PEGthat will inhibit the sample from running properly on the BioAnalyzer.

3. The remainder of the ligation reaction (190 μL) was used for gelisolation in Step 4.

Step 3a: Microcon Filtration and Adaptor Construction. Total PreparationTime was approximately 25 min.

The Universal Adaptor ligation reaction requires a 100-fold excess ofadaptors. To aid in the removal of these excess adaptors, thedouble-stranded gDNA library is filtered through a Microcon YM-100filter device. Microcon YM-100 membranes can be used to remove doublestranded DNA smaller than 125 bp. Therefore, unbound adaptors (44 bp),as well as adaptor dimers (88 bp) can be removed from the ligated gDNAlibrary population. The following filtration protocol was used:

1. The 190 μL of the ligation reaction from Step 4 was applied into anassembled Microcon YM-100 device.

2. The device was placed in a centrifuge and spun at 5000×g forapproximately 6 minutes, or until membrane was almost dry.

3. To wash, 200 μl of 1×TE was added.

4. Sample was spun at 5000×g for an additional 9 minutes, or untilmembrane was almost dry.

5. To recover, the reservoir was inserted into a new vial and spun at3000×g for 3 minutes. The reservoir was discarded. The recovered volumewas approximately 10 μl. Next, 80 μl TE was added.

The Adaptors (A and B) were HPLC-purified and modified withphosphorothioate linkages prior to use. For Adaptor “A” (10 μM), 10 μlof 100 μM Adaptor A (44 bp, sense) was mixed with 10 μl of 100 μMAdaptor A (40 bp, antisense), and 30 μl of 1× Annealing Buffer (V_(f)=50μl) were mixed. The primers were annealed using the ANNEAL program onthe Sample Prep Labthermal cycler (see below). For Adaptor “B” (10 μM),10 μl of 100 μM Adaptor B (40 bp, sense) was mixed with 10 μl of 100 μMAdaptor B (44 bp, antisense), and 30 μl of 1× Annealing Buffer (V_(f)=50μl). The primers were annealed using the ANNEAL program on the SamplePrep Lab thermal cycler. Adaptor sets could be stored at −20° C. untiluse.

ANNEAL-A program for primer annealing:

-   -   1. Incubate at 95° C., 1 min;    -   2. Decrease temperature to 15° C., at 0.1° C./sec; and    -   3. Hold at 15° C.

There was no orientation required for the genomic DNA insert fragmentand the adaptors. Fragments could be ligated at either end. Foursingle-stranded DNA oligonucleotides were included in the UniversalAdaptor set. Each single-stranded oligonucleotide was synthesized at 1micromole scale and HPLC-purified. Each single-stranded oligonucleotideincluded four phosphorothioate linkages at each end.

Step 4: Gel Electrophoresis and Extraction of Adapted DNA Library

The Universal Adaptor ligation protocol produces the following: 1)fragmented DNAs with adaptors on either end; 2) unbound single adaptors;or 3) the formation of adaptor dimers. Agarose gel electrophoresis isused as a method to separate and isolate the adapted DNA librarypopulation from the unligated, single adaptors and adaptor dimerpopulations. The procedure of DNase I digestion of genomic DNA yields alibrary population that ranges from 50-700 bp (Step 1). The addition ofthe 88-bp Universal Adaptor set will shift the population to a largersize and will result in a migration profile in the size range ofapproximately 130-800 bp. Adaptor dimers will migrate at 88 bp andadaptors unligated will migrate at 44 bp. Therefore, genomic DNAlibraries in size ranges >200 bp can be physically isolated from theagarose gel and purified using standard gel extraction techniques. Gelisolation of the adapted DNA library will result in the recovery of alibrary population in a size range that is >200 bp (size range oflibrary can be varied depending on application). The followingelectrophoresis and extraction protocol was used.

1. A 2% agarose gel was prepared.

2. 10 μl of 10× Ready-Load Dye was added to the remaining 90 μl of theDNA ligation mixture.

3. The dye/ligation reaction mixture was loaded into the gel using fouradjacent lanes (25 μl per lane).

4. 10 μl of the 100 bp ladder (0.1 μg/μl) was loaded two lanes away fromligation reaction lanes.

5. The gel was run at 100V for 3 hours.

6. When the gel run was complete, the gel was removed from the gel boxand transferred to a flat surface covered with plastic wrap. DNA bandswere visualized using a hand-held long-wave UV light. Using a sterile,single-use scalpel, the fragment sizes of 200-400 bp were cut out fromthe agarose gel. Using this approach, libraries with any size range canbe isolated. It is also possible to isolate more than one size range.Where the library size range is 200-900 bp, it is possible to isolateseveral size ranges from a single well (i.e., 200-400 bp and 500-700bp).

7. The DNA embedded in the agarose gel was isolated using a QiagenMinElute Gel Extraction kit following the manufacturer's instructions.Briefly, Buffer QG was added to cover the agarose in the tube. Theagarose was allowed to completely dissolve. The color of the Buffer QGwas maintained by adjusting the pH according to the Qiagen instructionsto minimize sample loss. Two MinElute spin columns (Qiagen) were usedfor purification. The large volume of dissolved agarose required eachcolumn to be loaded several times. The columns were eluded with 10 μl ofBuffer EB which was pre-warmed at 55° C. The eluates were pooled toproduce 20 μl of gDNA library.

8. One 1 μL aliquot of each isolated DNA library was analyzed using aBioAnalyzer DNA 1000 LabChip to assess the exact distribution of the DNAlibrary population.

Step 5: Strand Displacement and Extension of Nicked Double Stranded DNALibrary

Because the DNA oligonucleotides used for the Universal Adaptors are notphosphorylated, gaps are present at the 3′ junctions of the fragmentedgDNAs. These two “gaps” or “nicks” can be filled in by using a stranddisplacing DNA polymerase. The polymerase recognizes nicks, displacesthe nicked strands, and extends the strand in a manner that results inrepair of nicks and in the formation of non-nicked double-stranded DNA.The strand displacing enzyme used is the large fragment of Bst DNApolymerase.

1. In a 0.2 ml tube, 19 μl gel-extracted DNA library, 40 μl nH₂O, 8 μl10× ThermoPol Reaction Buffer, 8 μl BSA (1 mg/ml), 2 μl dNTPs (10 mM),and 3 μl Bst I Polymerase (8 U/μl) were added in order.

2. The samples were mixed well and placed in a thermal cycler andincubated using the Strand Displacement incubation program: “BST”. BSTprogram for stand displacement and extension of nicked double-strandedDNA:

-   -   1. Incubate at 65° C., 30 minutes;    -   2. Incubate at 80° C., 10 minutes;    -   3. Incubate at 58° C., 10 minutes; and    -   4. Hold at 14° C.

3. One 1 μL aliquot of the Bst-treated DNA library was run using aBioAnalyzer DNA 1000 LabChip.

Step 6: Preparation of Streptavidin Beads

Following the generation of unnicked double-stranded genomic DNA, it isnecessary to isolate single-stranded genomic DNAs containing flankingUniversal Adaptor sequences. This step outlines the binding ofbiotin-tagged double-stranded DNA to streptavidin beads. For preparingstreptavidin beads, the following protocol was used.

1. 100 μl Dynal M-270 Streptavidin beads were washed two times with 200μl of 1× Binding Buffer (1 M NaCl, 0.5 mM EDTA, 5 mM Tris, pH 7.5) byapplying the magnetic beads to the MPC.

2. The beads were resuspended in 100 μl 2× Binding buffer, then theremaining 79 μl of the Bst-treated DNA sample (from Step 5) and 20 μlwater was added.

3. The bead solution was mixed well and placed on a tube rotator at RTfor 20 minutes. The bead mixtures were washed, using the MPC, two timeswith 100 μl of 1× Binding Buffer, then washed two times with nH₂O.Binding & Washing (B&W) Buffer (2× and 1×): 2× B&W buffer was preparedby mixing 10 mM Tris.HCl (pH 7.5), 1 mM EDTA, and 2 M NaCl. The reagentswere combined as listed above and mixed thoroughly. The solution can bestored at RT for 6 months; 1× B&W buffer was prepared by mixing 2× B&Wbuffer with nH₂O, 1:1. The final concentrations were half the above,i.e., 5 mM Tris.HCl (pH 7.5), 0.5 mM EDTA, and 1 M NaCl.

Step 7: Isolation of single-stranded DNA Library using StreptavidinBeads

Following binding of the double-stranded gDNA library to streptavidinbeads, it is preferred to isolate from the ligated pool only thesingle-stranded gDNAs containing Universal Adaptor A and UniversalAdaptor B (desired populations are designated below with asterisks).Double-stranded genomic DNA fragment pools will have adaptors bound inthe following possible configurations:

Universal Adaptor A—gDNA Fragment—Universal Adaptor A

Universal Adaptor B—gDNA Fragment—Universal Adaptor A*

Universal Adaptor A—gDNA Fragment—Universal Adaptor B*

Universal Adaptor B—gDNA Fragment—Universal Adaptor B

Because only the Universal Adaptor B has a 5′ biotin moiety, magneticstreptavidin-containing beads can be used to bind all gDNA libraryspecies that possess the Universal Adaptor B. Genomic librarypopulations that contain two Universal Adaptor A species (or nonligatedspecies) do not bind to streptavidin-containing beads and are removedduring the wash procedure. The species that remain bound to bead afterwashing include those with Universal Adaptors A and B or those with twoUniversal Adaptor B ends.

Genomic DNA species with two Universal Adaptor B sequences with twobiotin molecules can bind to the streptavidin-containing beads at bothends. Species with A and B adaptors having only a single biotin moleculecan bind to the beads only at the “B” end. To isolate thesingle-stranded population, the bead-bound double-stranded DNA istreated with a sodium hydroxide solution that serves to disrupt thehydrogen bonding between the complementary DNA strands. If the DNAfragment has biotin on each end (Universal Adaptor B ends), bothresulting single strands remain bound to the beads. If the fragment hasonly a single biotin (Universal Adaptors A and B), then thecomplementary strand separates from the DNA-bead complex.

The resulting single-stranded genomic DNA library is collected from thesolution phase and is quantitated, e.g., using pyrophosphate sequencing(PyroSequence) or by using a RNA Pico 6000 LabChip (Agilent, Palo Alto,Calif.). Single-stranded genomic DNA libraries are quantitated bycalculating the number of molecules per unit volume. Single-strandedgDNA molecules are then annealed (at a half copy per bead to obtain oneeffective copy per bead) to 25-30 μm sepharose beads containing DNAcapture primers (PCR primer B). The templates are then amplified usingemulsion polymerase chain reaction protocols. Subsequent sequencing maybe conducted using known techniques. For isolation of the singlestranded library, the following protocol was used.

1. 250 μl Melt Solution (0.125 M NaOH, 0.1 M NaCl) was added to washedbeads from Step 6 above.

2. The bead solution was mixed well and the bead mixture was incubatedat room temperature for 10 minutes on a tube rotator.

3. A Dynal MPC (magnetic particle concentrator) was used, the pelletbeads were carefully removed, and the supernatant was set aside. The250-μl supernatant included the single-stranded DNA library.

4. In a separate tube, 1250 μl PB (from QiaQuick Purification kit) wasadded and the solution was neutralized by adding 9 μl of 20% aceticacid.

5. Using a Dynal MPC, beads from the 250-μl supernatant including thesingle-stranded gDNA library were pelleted and the supernatant wascarefully removed and transferred to the freshly prepared PB/acetic acidsolution.

6. The 1500 μl solution was purified using a single QiaQuickpurification spin column (load sample through same column two times at750 μl per load). The single-stranded DNA library was eluted with 50 μlEB.

Step 8a: Single-stranded gDNA Quantitation using PyrophosphateSequencing. Total Preparation Time was Approximately 1 hr.

1. In a 0.2 ml tube, the following reagents were added in order:

-   -   25 μl single-stranded gDNA    -   1 μl MMP2B sequencing primer    -   14 μl Library Annealing Buffer    -   40 μl total

2. The DNA was allowed to anneal using the ANNEAL-S Program (seeAppendix, below).

3. The samples were run on PSQ (pyrophosphate sequencing jig) todetermine the number of picomoles of template in each sample (seebelow). Methods of sequencing can be found in U.S. Pat. No. 6,274,320;U.S. Pat. No. 4,863,849; U.S. Pat. No. 6,210,891; and U.S. Pat. No.6,258,568, the disclosures of which are incorporated in toto herein byreference. Calculations were performed to determine the number ofsingle-stranded gDNA template molecules per microliter. The remaining 25μL of prepared single-stranded gDNA library was used for amplificationand subsequent sequencing (approximately 1×10⁶ reactions).

Step 8b: Single-Stranded gDNA Quantitation using RNA Pico 6000 LabChip.Total Preparation Time was Approximately 30 minutes.

1. The mRNA Pico assay option was selected on the BioAnalyzer (Softwareversion 2.12).

2. An RNA Pico 6000 LabChip was prepared on the BioAnalyzer according tothe manufacturers' guidelines.

3. An RNA LabChip ladder (RNA 6000 ladder) was prepared according tomanufacturer's (Ambion) directions. Briefly, the RNA LabChip ladder, insolution, was heated to 70° C. for 2 minutes. The solution was chilledon ice for 5 minutes to snap cool the ladder. The solution was brieflycentrifuged to clear any condensate from tube walls. The RNA LabChipLadder was stored on ice and used within one day.

4. The ssDNA library to be analyzed was run in triplicate, in adjacentlanes, using three 1 μl aliquots.

5. The BioAnalyzer software was used to calculate the concentration ofeach ssDNA library lane (see the Table below and FIG. 24. The average ofall three lanes was used to calculate the DNA concentration of thelibrary using the procedure outlined below.

-   -   a. The peak integration lower limit line (large dash in FIG. 24)        was moved immediately in front of the library peak (see below).    -   b. The peak integration upper limit line (large dash in the        FIG. 24) was moved immediately after the library peak. In this        way, the peak integration line connecting the lower and upper        integration lines followed the slope of the background.    -   c. The mouse arrow was used to determine the average size of the        peak in bases (usually near the peaks highest point) or a        defined peak was used as chosen by the software.    -   d. The integrated value was used for the amount of material in        the peak. The value obtained for picograms recovered was        converted into molecules recovered (see Table, below). The        library concentration was then determined (molecules per        microliter).

TABLE 5 6 7 8 9 2 3 4 Average Mean Mean Mean Average 1 pg/μL (1) pg/μL(2) pg/μL (3) pg/μL Size (bp) 1 Size (bp) 2 Size (bp) 3 Size (bp) sample1633 1639 1645 1639 435 435 432 434 10 11 12 Ave MW (g/mole) Ave MWLibrary 13 14 15 1 Ribonucleotide (g/mole) g/μL moles/g moles/μLmolecules/μL sample 328.2 1.42E+05 1.64E−09 7.02E−06 1.15E−14 6.93E+09

As shown in the Table above, the concentration of Library 1 wascalculated as 1639 pg/μl (Column 5) and the average fragment size was434 nucleotides (Column 9). These values were obtained from the Agilent2100 software as described in Steps (a)-(d), above. The averagemolecular weight (MW) of a ribonucleotide is 328.2 g/mole (Column 10).The MW of the average library fragment (1.42×10⁵ g/mole, Column 11) wascalculated by multiplying the average fragment length (434) by theaverage ribonucleotide (328.2). The quantitated library (1639 pg/μl) wasconverted to grams per microliter (1.64×10⁻⁹ g/μl, Column 12). Thenumber of moles per microliter (1.15×10⁻¹⁴ moles/μl, Column 14) wascalculated by dividing the grams per microliter (1.64×10⁻⁹ g/μl, Column12) by the average molecular weight of the library fragments (1.42×10⁵,Column 11). Finally, the number of molecules per microliter (6.93×10⁹molecules/μl, Column 15) was derived by multiplying the number of molesper microliter (1.15×10⁻¹⁴ moles/μl, Column 14) by Avogadro's number(6.02×10²³ molecules/mole).

The final library concentration was expected to be greater than 1×10⁸molecules/μl. A more important factor for library quality was adaptordimer concentration. In FIG. 24, the height of the library peak wasdetermined approximately 10 fold greater than the adaptor dimer peak(the first peak after the marker). A library of good quality is expectedto have a peak height at least 2 fold greater than the dimer peak. Itshould be noted that the RNA Pico 6000 LabChip provided estimates within500% accuracy of the single-stranded gDNA concentration. Thus, it wasimportant to perform an initial sequencing run using a titration oftemplate to determine the number of copies per bead (cpb) of input gDNA.The recommended input DNA is 2.5 cpb, 1 cpb, 0.5 cpb, and 0.1 cpb. Thistitration was easily checked using the 4slot bead loading chamber on a14×43 PTP.

Step 9: Dilution and Storage of Single-Stranded gDNA Library

The single-stranded gDNA library was eluted and quantitated in BufferEB. To prevent degradation, the single-stranded gDNA library was storedfrozen at −20° C. in the presence of EDTA. After quantitation, an equalvolume of 10 mM TE was added to the library stock. All subsequentdilutions was in TE. The yield was as follows:

Remaining final volume of ssDNA library following PSQ analysis=25 μl.

Remaining final volume of ssDNA library following LabChip analysis=47μl.

For the initial stock dilution, single-stranded gDNA library was dilutedto 100 million molecules/μl in 1× Library-Grade Elution Buffer. Aliquotsof single-stranded gDNA library were prepared for common use. For this,200,000 molecules/μl were diluted in 1× Library-Grade Elution Buffer and20 μl aliquots were measured. Single-use library aliquots were stored at−20° C.

Step 10: Emulsion Polymerase Chain Reaction

Where increased numbers of cpb were preferred, bead emulsion PCR wasperformed as described in U.S. Patent Application Serial No. 06/476,504filed Jun. 6, 2003, incorporated herein by reference in its entirety.

Reagent Preparation

The Stop Solution (50 mM EDTA) included 100 μl of 0.5 M EDTA mixed with900 μl of nH₂O to obtain 1.0 ml of 50 mM EDTA solution. For 10 mM dNTPs,(10 μl dCTP (100 mM), 10 μl dATP (100 mM), 10 μl dGTP (100 mM), and 110μl dTTP (100 mM) were mixed with 60 μl molecular biology grade water.All four 100 mM nucleotide stocks were thawed on ice. Then, 10 μl ofeach nucleotide was combined with 60 μl of nH₂O to a final volume of 100μl, and mixed thoroughly. Next, 1 ml aliquots were dispensed into 1.5 mlmicrocentrifuge tubes. The stock solutions could be stored at −20° C.for one year.

The 10× Annealing buffer included 200 mM Tris (pH 7.5) and 50 mMmagnesium acetate. For this solution, 24.23 g Tris was added to 800 mlnH₂O and the mixture was adjusted to pH 7.5. To this solution, 10.72 gof magnesium acetate was added and dissolved completely. The solutionwas brought up to a final volume of 1000 ml and could be stored at 4° C.for 1 month. The 10× TE included 100 mM Tris.HCl (pH 7.5) and 50 mMEDTA. These reagents were added together and mixed thoroughly. Thesolution could be stored at room temperature for 6 months.

Example 2 Primer Design

As discussed above, the universal adaptors are designed to include: 1) aset of unique PCR priming regions that are typically 20 bp in length(located adjacent to (2)); 2) a set of unique sequencing priming regionsthat are typically 20 bp in length; and 3) optionally followed by aunique discriminating key sequence consisting of at least one of each ofthe four deoxyribonucleotides (i.e., A, C, G, T). The probability ofcross-hybridization between primers and unintended regions of the genomeof interest is increased as the genome size increases and length of aperfect match with the primer decreases. However, this potentialinteraction with a cross-hybridizing region (CHR) is not expected toproduce problems for the reasons set forth below.

In a preferred embodiment of the present invention, the single-strandedDNA library is utilized for PCR amplification and subsequent sequencing.Sequencing methodology requires random digestion of a given genome into150 to 500 base pair fragments, after which two unique bipartite primers(composed of both a PCR and sequencing region) are ligated onto the 5′and 3′ ends of the fragments (FIG. 25). Unlike typical PCRamplifications where an existing section of the genome is chosen as apriming site based on melting temperature (T_(m)), uniqueness of thepriming sequence within the genome and proximity to the particularregion or gene of interest, the disclosed process utilizes syntheticpriming sites that necessitates careful de novo primer design.

Tetramer Selection:

Strategies for de novo primer design are found in the publishedliterature regarding work conducted on molecular tags for hybridizationexperiments (see, Hensel, M. and D. W. Holden, Molecular geneticapproaches for the study of virulence in both pathogenic bacteria andfungi. Microbiology, 1996. 142(Pt 5): p. 1049-58; Shoemaker, D. D., etal., Quantitative phenotypic analysis of yeast deletion mutants using ahighly parallel molecular bar-coding strategy. Nat Genet, 1996. 14(4):p. 450-6) and PCR/LDR (polymerase chain reaction/ligation detectionreaction) hybridization primers (see, Gerry, N. P., et al., UniversalDNA microarray method for multiplex detection of low abundance pointmutations. Journal of Molecular Biology, 1999. 292: p. 251-262;Witowski, N. E., et al., Microarray-based detection of selectcardiovascular disease markers. BioTechniques, 2000. 29(5): p.936-944.).

The PCR/LDR work was particularly relevant and focused on designingoligonucleotide “zipcodes”, 24 base primers comprised of sixspecifically designed tetramers with a similar final T_(m). (see, Gerry,N. P., et al., Universal DNA microarray method for multiplex detectionof low abundance point mutations. Journal of Molecular Biology, 1999.292: p. 251-262; U.S. Pat. No. 6,506,594). Tetrameric components werechosen based on the following criteria: each tetramer differed from theothers by at least two bases, tetramers that induced self-pairing orhairpin formations were excluded, and palindromic (AGCT) or repetitivetetramers (TATA) were omitted as well. Thirty-six of the 256 (4⁴)possible permutations met the necessary requirements and were thensubjected to further restrictions required for acceptable PCR primerdesign (Table 1).

TABLE 1

The table shows a matrix demonstrating tetrameric primer componentselection based on criteria outlined by Gerry et al. 1999. J. Mol. Bio.292: 251-262. Each tetramer was required to differ from all others by atleast two bases. The tetramers could not be palindromic or complimentarywith any other tetramer. Thirty-six tetramers were selected (bold,underlined); italicized sequences signal palindromic tetramers that wereexcluded from consideration.

Primer Design:

The PCR primers were designed to meet specifications common to generalprimer design (see, Rubin, E. and A. A. Levy, A mathematical model and acomputerized simulation of PCR using complex templates. Nucleic AcidsRes, 1996. 24(18): p. 3538-45; Buck, G. A., et al., Design strategiesand performance of custom DNA sequencing primers. Biotechniques, 1999.27(3): p. 528-36), and the actual selection was conducted by a computerprogram, MMP. Primers were limited to a length of 20 bases (5 tetramers)for efficient synthesis of the total bipartite PCR/sequencing primer.Each primer contained a two base GC clamp on the 5′ end, and a single GCclamp on the 3′ end (Table 2), and all primers shared similar T_(m)(+/−2° C.) (FIG. 27). No hairpinning within the primer (internal hairpinstem ΔG>−1.9 kcal/mol) was permitted. Dimerization was also controlled;a 3 base maximum acceptable dimer was allowed, but it could occur infinal six 3′ bases, and the maximum allowable ΔG for a 3′ dimer was −2.0kcal/mol. Additionally, a penalty was applied to primers in which the 3′ends were too similar to others in the group, thus preventingcross-hybridization between one primer and the reverse complement ofanother.

TABLE 2 1-pos 2-pos 3-pos 4-pos 5-pos  1 CCAT TGAT TGAT TGAT ATAC  2CCTA CTCA CTCA CTCA AAAG  3 CGAA TACA TACA TACA TTAG  4 CGTT AGCC AGCCAGCC AATC  5 GCAA GACC GACC GACC TGTC  6 GCTT TCCC TCCC TCCC AGTG  7GGAC ATCG ATCG ATCG CTTG  8 GGTA CACG CACG CACG GATG  9 TGCG TGCG TGCGTCTG 10 ACCT ACCT ACCT 11 GTCT GTCT GTCT 12 AGGA AGGA AGGA 13 TTGA TTGATTGA 14 CAGC CAGC CAGC 15 GTGC GTGC GTGC 16 ACGG ACGG ACGG 17 CTGT CTGTCTGT 18 GAGT GAGT GAGT 19 TCGT TCGT TCGT

Table 2 shows possible permutations of the 36 selected tetrads providingtwo 5′ and a single 3′ G/C clamp. The internal positions are composed ofremaining tetrads. This results in 8×19×19×19×9 permutations, or 493,848possible combinations. FIG. 27 shows first pass, T_(m) based selectionof acceptable primers, reducing field of 493,848 primers to 56,246candidates with T_(m) of 64 to 66° C.

TABLE 3 The probability of perfect sequence matches for primersincreases with decreasing match length requirements and increasing sizeof the genome of interest. % chance for match Perfect % chance in NCBImatch for match bacterial % chance for Match probability in Adenodatabase match in Human Length (1/(4{circumflex over ( )}length)) ~35Kbases ~488 M bases ~3 B bases 20 9.1E−13 0.00% 0.04% 0.27% 19 7.3E−120.00% 0.65% 4.32% 18 4.4E−11 0.00% 5.76% 34.37%  17 2.3E−10 0.00%35.69%  99.17%  16 1.2E−09 0.02% 97.52%  >100%  15 5.6E−090.12% >100%  >100%  14 2.6E−08 0.64% >100%  >100%  13 1.2E−073.29% >100%  >100%  12 5.4E−07 15.68%  >100%  >100%  11 2.4E−0658.16%  >100%  >100%  10 1.0E−05 99.35%  >100%  >100%  9 4.6E−0599.77%  >100%  >100%  8 2.0E−04 >100%  >100%  >100%  78.5E−04 >100%  >100%  >100%  6 3.7E−03 >100%  >100%  >100%  51.6E−02 >100%  >100%  >100%  4 6.4E−02 >100%  >100%  >100%  32.5E−01 >100%  >100%  >100%  2 7.1E−01 >100%  >100%  >100%  11.0E+00 >100%  >100%  >100% 

The possibility of complimentary regions occurring within the genome ofinterest was not a major concern in the primer design process despitethe reported tolerance of PCR to mismatches in complex samplepopulations (see, e.g., Rubin, E. and A. A. Levy, A mathematical modeland a computerized simulation of PCR using complex templates. NucleicAcids Res, 1996. 24(18): p. 3538-45). Although the probability offinding a perfect match to a 20 base primer is extremely low (4²⁰)(Table 3), the probability of finding less non-consecutive matchesincreases significantly with the size of the genome of interest. As aresult, the probability of finding a perfect match of at least 10 of 20bases is 99.35% for an Adenovirus genome. The probability of finding a16 base perfect match is 97% for the sequences in the NCBI database(approximately 100 times larger than the Adenovirus genome). Theprobability of finding a 17 base perfect match to a 20 base primer is99% for the sequences in the human genome (3 billion bases).

The high probability of primer cross-hybridization to regions of thegenome is less problematic than one might expect due to the random DNAdigestion used to produce the template fragments. Thus, the effects of across-hybridizing region (CHR) are fairly benign. It is unlikely that aCHR would be able to successfully compete with the perfect match betweenthe PCR primers in solution and the template. In addition, any primersthat include mismatches at their 3′ end would be at a significantcompetitive disadvantage. Even if a CHR should out compete the intendedPCR primer, it would produce a truncated PCR product, without adownstream site for the sequencing primer. If the truncated productcould be driven to the capture bead and immobilized, one of twosituations would result. If the CHR out-competed the solution-phaseprimer, then the immobilized product would lack a sequencing primerbinding site, and would result in an empty PicoTiter plate (PTP) well.If the CHR out-competed the bead-bound primer, the sequencing primerwould still be present, and the only effect would be a shorter insert.Neither result would unduly compromise the sequencing quality. Given thelarge amount of genomic material used in the sample preparation process(currently 25 μg, containing 5.29×10¹⁶ copies of the 35 Kb Adenovirusgenome), oversampling can be used to provide fragments that lack thecomplete CHR, and allow standard PCR amplification of the region inquestion.

Example 3 Sample Preparation by Nebulization

Preparation of DNA by Nebulization

The purpose of the Nebulization step is to fragment a large stretch ofDNA such as a whole genome or a large portion of a genome into smallermolecular species that are amenable to DNA sequencing. This populationof smaller-sized DNA species generated from a single DNA template isreferred to as a library. Nebulization shears double-stranded templateDNA into fragments ranging from 50 to 900 base pairs. The shearedlibrary contains single-stranded ends that are end-repaired by acombination of T4 DNA polymerase, E. coli DNA polymerase I (Klenowfragment), and T4 polynucleotide kinase. Both T4 and Klenow DNApolymerases are used to “fill-in” 3′ recessed ends (5′ overhangs) of DNAvia their 5′-3′ polymerase activity. The single-stranded 3′-5′exonuclease activity of T4 and Klenow polymerases will remove 3′overhang ends and the kinase activity of T4 polynucleotide kinase willadd phosphates to 5′ hydroxyl termini.

The sample was prepared as follows:

1. 15 μg of gDNA (genomic DNA) was obtained and adjusted to a finalvolume of 100 μl in 10 mM TE (10 mM Tris, 0.1 mM EDTA, pH 7.6; seereagent list at the end of section). The DNA was analyzed forcontamination by measuring the O.D. 260/280 ratio, which was 1.8 orhigher. The final gDNA concentration was expected to be approximately300 μg/ml.

2. 1600 μl of ice-cold Nebulization Buffer (see end of section) wasadded to the gDNA.

3. The reaction mixture was placed in an ice-cold nebulizer (CIS-US,Bedford, Mass.).

4. The cap from a 15 ml snap cap falcon tube was placed over the top ofthe nebulizer (FIG. 28A).

5. The cap was secured with a clean Nebulizer Clamp assembly, consistingof the fitted cover (for the falcon tube lid) and two rubber O-rings(FIG. 28B).

6. The bottom of the nebulizer was attached to a nitrogen supply and theentire device was wrapped in parafilm (FIGS. 28C and 28D).

7. While maintaining nebulizer upright (as shown in FIG. 28D), 50 psi(pounds per square inch) of nitrogen was applied for 5 minutes. Thebottom of the nebulizer was tapped on a hard surface every few secondsto force condensed liquid to the bottom.

8. Nitrogen was turned off after 5 minutes. After the pressure hadnormalized (30 seconds), the nitrogen source was remove from thenebulizer.

9. The parafilm was removed and the nebulizer top was unscrewed. Thesample was removed and transferred to a 1.5 ml microcentrifuge tube.

10. The nebulizer top was reinstalled and the nebulizer was centrifugedat 500 rpm for 5 minutes.

11. The remainder of the sample in the nebulizer was collected. Totalrecovery was about 700 μl.

12. The recovered sample was purified using a QIAquick column (QiagenInc., Valencia, Calif.) according to manufacturer's directions. Thelarge volume required the column to be loaded several times. The samplewas eluted with 30 μl of Buffer EB (10 mM Tris HCl, pH 8.5; supplied inQiagen kit) which was pre-warmed at 55° C.

13. The sample was quantitated by UV spectroscopy (2 μl in 198 μl waterfor 1:100 dilution).

Enzymatic Polishing

Nebulization of DNA templates yields many fragments of DNA with frayedends. These ends are made blunt and ready for ligation to adaptorfragments by using three enzymes, T4 DNA polymerase, E. coli DNApolymerase (Klenow fragment) and T4 polynucleotide kinase.

The sample was prepared as follows:

1. In a 0.2 ml tube the following reagents were added in order:

-   -   28 μl purified, nebulized gDNA fragments    -   5 μl water    -   5 μl 10× T4 DNA polymerase buffer    -   5 μl BSA (1 mg/ml)    -   2 μl dNTPs (10 mM)    -   5 μl T4 DNA polymerase (3 units/μl)    -   50 μl final volume

2. The solution of step 1 was mixed well and incubated at 25° C. for 10minutes in a MJ thermocycler (any accurate incubator may be used).

3. 1.25 μl E. coli DNA polymerase (Klenow fragment) (5 units/ml) wasadded.

4. The reaction was mixed well and incubated in the MJ thermocycler for10 minutes at 25° C. and for an additional 2 hrs at 16° C.

5. The treated DNA was purified using a QiaQuick column and eluted with30 μl of Buffer EB (10 mM Tris HCl, pH 8.5) which was pre-warmed at 55°C.

6. The following reagents were combined in a 0.2 ml tube:

-   -   30 μl Qiagen purified, polished, nebulized gDNA fragments    -   5 μl water    -   5 μl 10× T4 PNK buffer    -   5 μl ATP (10 mM)    -   5 μl T4 PNK (10 units/ml)    -   50 μl final volume

7. The solution was mixed and placed in a MJ thermal cycler using the T4PNK program for incubation at 37° C. for 30 minutes, 65° C. for 20minutes, followed by storage at 14° C.

8. The sample was purified using a QiaQuick column and eluted in 30 μlof Buffer EB which was pre-warmed at 55° C.

9. A 2 μl aliquot of the final polishing reaction was held for analysisusing a BioAnalyzer DNA 1000 LabChip (see below).

Ligation of Adaptors

The procedure for ligating the adaptors was performed as follows:

1. In a 0.2 ml tube the following reagents were added in order:

-   -   20.6 μl molecular biology grade water    -   28 μl digested, polished gDNA Library    -   60 μl 2× Quick Ligase Reaction Buffer    -   1.8 μl MMP (200 pmol/μl) Universal Adaptor set    -   9.6 μl Quick Ligase    -   120 μl total

The above reaction was designed for 5 μg and was scaled depending on theamount of gDNA used.

2. The reagents were mixed well and incubated at 25° C. for 20 minutes.The tube was on ice until the gel was prepared for agarose gelelectrophoresis.

Gel Electrophoresis and Extraction of Adapted gDNA Library

Nebulization of genomic DNA yields a library population that ranges from50-900 bp. The addition of the 88-bp Universal Adaptor set will shiftthe population to a larger size and will result in a migration profilewith a larger size range (approximately 130-980 bp). Adaptor dimers willmigrate at 88 bp and adaptors not ligated will migrate at 44 bp.Therefore, genomic DNA libraries isolated in size ranges >250 bp can bephysically isolated from the agarose gel and purified using standard gelextraction techniques. Gel isolation of the adapted gDNA library willresult in the recovery of a library population in a size range thatis >250 bp (size range of library can be varied depending onapplication). The library size range after ligation of adapters is 130to 980 bp. It should be noted that the procedure may be adapted forisolation of any band size range, such as, for example, 130 to 200 bp,200 to 400 bp, 250 to 500 bp, 300 to 600 bp, 500 to 700 bp and the likeby cutting different regions of the gel. The procedure described belowwas used to isolated fragments of 250 bp to 500 bp.

A 150 ml agarose gel was prepared to include 2% agarose, 1×TBE, and 4.5μl ethidium bromide (10 mg/ml stock). The ligated DNA was mixed with 10×Ready Load Dye and loaded onto the gel. In addition, 10 μl of a 100-bpladder (0.1 μg/μl) was loaded on two lanes away from the ligationreaction flanking the sample. The gel was electrophoresed at 100 V for 3hours. When the gel run was complete, the gel was removed from the gelbox, transferred to a GelDoc, and covered with plastic wrap. The DNAbands were visualized using the Prep UV light. A sterile, single-usescalpel, was used to cut out a library population from the agarose gelwith fragment sizes of 250-500 bp. This process was done as quickly aspossible to prevent nicking of DNA. The gel slices were placed in a 15ml falcon tube. The agarose-embedded gDNA library was isolated using aQiagen MinElute Gel Extraction kit. Aliquots of each isolated gDNAlibrary were analyzed using a BioAnalyzer DNA 1000 LabChip to assess theexact distribution of the gDNA library population.

Strand Displacement and Extension of the gDNA Library and Isolation ofthe Single Stranded gDNA Library Using Streptavidin Beads

Strand displacement and extension of nicked double-stranded gDNA librarywas performed as described in Example 1, with the exception that theBst-treated samples were incubated in the thermal cycler at 65° C. for30 minutes and placed on ice until needed. Streptavidin beads wereprepared as described in Example 1, except that the final wash wasperformed using two washes with 200 μl 1× Binding buffer and two washeswith 200 μl nH₂O, Single-stranded gDNA library was isolated usingstreptavidin beads as follows. Water from the washed beads was removedand 250 μl of Melt Solution (see below) was added. The bead suspensionwas mixed well and incubated at room temperature for 10 minutes on atube rotator. In a separate tube, 1250 μl of PB (from the QiaQuickPurification kit) and 9 μl of 20% acetic acid were mixed. The beads in250 μl Melt Solution were pelleted using a Dynal MPC and the supernatantwas carefully removed and transferred to the freshly prepared PB/aceticacid solution. DNA from the 1500 μl solution was purified using a singleMinElute purification spin column. This was performed by loading thesample through the same column twice at 750 μl per load. The singlestranded gDNA library was eluted with 15 μl of Buffer EB which waspre-warmed at 55° C.

Single Strand gDNA Quantitation and Storage

Single-stranded gDNA was quantitated using RNA Pico 6000 LabChip asdescribed in Example 1. In some cases, the single stranded library wasquantitated by a second assay to ensure the initial Agilent 2100quantitation was performed accurately. For this purpose, RiboGreenquantitation was performed as described (ssDNA Quantitation byFluorometry) to confirm the Agilent 2100 quantitation. If the twoestimates differed by more than 3 fold, each analysis was repeated. Ifthe quantitation showed greater than a 3 fold difference between the twoprocedures, a broader range of template to bead was used.

Dilution and storage of the single stranded gDNA library was performedas described in Example 1. The yield was as follows:

Remaining final volume of ssDNA library following LabChip analysis=12μl.

Remaining final volume of ssDNA library following RiboGreen analysis=9μl.

Final volume of ssDNA library after the addition of TE=18 μl.

An equal volume of TE was added to single-stranded gDNA library stock.Single-stranded gDNA library to 1×10⁸ molecules/μl in Buffer TE. Stockwas diluted (1/500) to 200,000 molecules/μl in TE and 20 μl aliquotswere prepared.

Library Fragment Size Distribution After Nebulization

Typical results from Agilent 2100 DNA 1000 LabChip analysis of 1 μl ofthe material following Nebulization and polishing are shown in FIG. 29A.The size range distribution of the majority of the product was expectedto fall around 50 to 900 base pairs. The mean size (top of peak) wasexpected to be approximately 450 bp. Typical results from gelpurification of adaptor ligated library fragments are shown in FIG. 29B.

Reagents

Unless otherwise specified, the reagents listed in the Examplesrepresent standard reagents that are commercially available. Forexample, Klenow, T4 DNA polymerase, T4 DNA polymerase buffer, T4 PNK, T4PNK buffer, Quick T4 DNA Ligase, Quick Ligation Buffer, Bst DNApolymerase (Large Fragment) and ThermoPol reaction buffer are availablefrom New England Biolabs (Beverly, Mass.). dNTP mix is available fromPierce (Rockford, Ill.). Agarose, UltraPure TBE, BlueJuice gel loadingbuffer and Ready-Load 100 bp DNA ladder may be purchased from Invitrogen(Carlsbad, Calif.). Ethidium Bromide and 2-Propanol may be purchasedfrom Fisher (Hampton, N.H.). RNA Ladder may be purchased from Ambion(Austin, Tex.). Other reagents are either commonly known and/or arelisted below:

Melt Solution:

Ingredient Quantity Required Vendor Stock Number NaCl (5 M)   200 μlInvitrogen 24740-011 NaOH (10 N)   125 μl Fisher SS255-1 molecularbiology 9.675 ml Eppendorf 0032-006-205 grade water

The Melt Solution included 100 mM NaCl, and 125 mM NaOH. The listedreagents were combined and mixed thoroughly. The solution could bestored at RT for six months.

Binding & Washing (B&W) Buffer (2× and 1×):

Ingredient Quantity Required Vendor Stock Number UltraPure Tris-HCl  250μl Invitrogen 15567-027 (pH 7.5, 1 M) EDTA (0.5 M)   50 μl Invitrogen15575-020 NaCl (5 M)   10 ml Invitrogen 24740-011 molecular biology 14.7ml Eppendorf 0032-006-205 grade water

The 2× B&W buffer included final concentrations of 10 mM Tris-HCl (pH7.5), 1 mM EDTA, and 2 M NaCl. The listed reagents were combined bycombined and mixed thoroughly. The solution could be stored at RT for 6months. The 1× B&W buffer was prepared by mixing 2× B&W buffer withpicopure H₂O, 1:1. The final concentrations was half of that listed theabove, i.e., 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, and 1 M NaCl.

Other buffers included the following. 1× T4 DNA Polymerase Buffer: 50 mMNaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol (pH 7.9 @ 25°C.). TE: 10 mM Tris, 1 mM EDTA.

Special Reagent Preparation:

TE (10 mM):

Ingredient Quantity Required Vendor Stock Number TE (1M)  1 ml FisherBP1338-1 molecular biology 99 ml Eppendorf 0032-006-205 grade water

Reagents were mixed and the solution could be stored RT for six months.

Nebulization Buffer:

Ingredient Quantity Required Vendor Stock Number Glycerol 53.1 ml SigmaG5516 molecular biology 42.1 ml Eppendorf 0032-006-205 grade waterUltraPure Tris-HCl  3.7 ml Invitrogen 15567-027 (pH 7.5, 1M) EDTA (0.5M) 1.1 ml Sigma M-10228

All reagents were added (glycerol was added last) to a Stericup andmixed well. The solution was labeled and could be stored at RT for sixmonths.

ATP (10 mM):

Ingredient Quantity Required Vendor Stock Number ATP (100 mM) 10 μlRoche 1140965 molecular biology 90 μl Eppendorf 0032-006-205 grade water

The reagents were mixed and the solution could be stored at −20° C. forsix months.

BSA (1 mg/ml):

Ingredient Quantity Required Vendor Stock Number BSA (10 mg/ml) 10 μlNEB M0203 kit Molecular Biology 90 μl Eppendorf 0032-006-205 Grade water

The reagents were mixed and the solution could be stored at 4° C. forsix months.

Library Annealing buffer, 10×:

Ingredient Quantity Req. Vendor Stock No. UltraPure Tris-HCl   200 mlInvitrogen 15567-027 (pH 7.5, 1 M) Magnesium acetate, 10.72 g FisherBP-215-500 enzyme grade (1 M) Molecular Biology   ~ 1 L Eppendorf0032-006-205 Grade water

The 10× Annealing Buffer included 200 mM Tris (pH 7.5) and 50 mMmagnesium acetate. For this buffer, 200 ml of Tris was added to 500 mlpicopure H₂O. Next, 10.72 g of magnesium acetate was added to thesolution and dissolved completely. The solution was adjusted to a finalvolume of 1000 ml. The solution could be stored at 4° C. for six months.To avoid the potential for contamination of libraries, the buffer wasaliquotted for single or short-term usage.

Adaptors:

Adaptor “A” (400 μM):

Ingredient Quantity Req. Vendor Stock No. Adaptor A (sense;, 10.0 μl IDTcustom HPLC-purified phosphorothioate linkages, 44 bp, 1000 pmol/μl)Adaptor A (antisense; 10.0 μl IDT custom HPLC-purified, Phosphorothioatelinkages, 40 bp, 1000 pmol/μl) Annealing buffer (10X)  2.5 μl 454 Corp.previous table molecular biology  2.5 μl Eppendorf 0032-006-205 gradewater

For this solution, 10 μl of 1000 μmol/μl Adaptor A (44 bp, sense) wasmixed with 10 μl of 1000 μmol/μl Adaptor A (40 bp, antisense), 2.5 μl of10× Library Annealing Buffer, and 2.5 μl of water (V_(f)=25 μl). Theadaptors were annealed using the ANNEAL-A program (see Appendix, below)on the Sample Prep Lab thermal cycler. More details on adaptor designare provided in the Appendix.

Adaptor “B” (400 μM):

Ingredient Quantity Req. Vendor Stock No. Adaptor B (sense;  10 μl IDTCustom HPLC-purified, phosphorothioate linkages, 40 bp, 1000 pmol/μl))Adaptor B (anti;  10 μl IDT Custom HPLC-purified, phosphorothioatelinkages, 5′Biotinylated, 44 bp, 1000 pmol/μl) Annealing buffer (10X)2.5 μl 454 Corp. previous table molecular biology 2.5 μl Eppendorf0032-006-205 grade water

For this solution, 10 μl of 1000 μmol/μl Adaptor B (40 bp, sense) wasmixed with 10 μl of 1000 μmol/μl Adaptor B (44 bp, anti), 2.5 μl of 10×Library Annealing Buffer, and 2.5 μl of water (V_(f)=25 μl). Theadaptors were annealed using the ANNEAL-A program (see Appendix) on theSample Prep Lab thermal cycler. After annealing, adaptor “A” and adaptor“B” (V_(f)=50 μl) were combined. Adaptor sets could be stored at −20° C.until use.

20% Acetic Acid:

Ingredient Quantity Required Vendor Stock Number acetic acid, glacial 2ml Fisher A35-500 molecular biology 8 ml Eppendorf 0032-006-205 gradewater

For this solution, glacial acetic acid was added to the water. Thesolution could be stored at RT for six months.

Adaptor Annealing Program:

ANNEAL-A program for primer annealing:

-   -   1. Incubate at 95° C., 1 min;    -   2. Reduce temperature to 15° C. at 0.1° C./sec; and    -   3. Hold at 14° C.        T4 Polymerase/Klenow POLISH program for end repair:    -   1. Incubate at 25° C., 10 minutes;    -   2. Incubate at 16° C., 2 hours; and    -   3. Hold at 4° C.        T4 PNK Program for End Repair:    -   1. Incubate at 37° C., 30 minutes;    -   2. Incubate at 65° C., 20 minutes; and    -   3. Hold at 14° C.        BST Program for Stand Displacement and Extension of Nicked        Double-Stranded gDNA:    -   1. Incubate at 65° C., 30 minutes; and    -   2. Hold at 14° C.        Step 9: Dilution and Storage of Single-Stranded DNA Library

Single-stranded DNA library in EB buffer: remaining final volume=25 μl.

Initial Stock dilution was made as follows. Using Pyrosequencing(Pyrosequencing AB, Uppsala, Sweden) results, single-stranded DNAlibrary was diluted to 100M molecules/μL in 1× Annealing Buffer (usuallythis was a 1:50 dilution).

Aliquots of single-stranded DNA Library were made for common use bydiluting 200,000 molecules/μL in 1× Annealing Buffer and preparing 30 μLaliquots. Store at −20° C. Samples were utilized in emulsion PCR.

Reagent Preparation:

Stop Solution (50 mM EDTA): 100 μl of 0.5 M EDTA was mixed with 900 μlof nH₂O to make 1.0 ml of 50 mM EDTA solution.

Solution of 10 mM dNTPs included 10 μl dCTP (100 mM), 101 dATP (100 mM),10 μl dGTP (100 mM), and 10 μl dTTP (100 mM), 60 μl Molecular BiologyGrade water, (nH₂O). All four 100 mM nucleotide stocks were thawed onice. 10 μl of each nucleotide was combined with 60 μl of nH₂O to a finalvolume of 100 μl, and mixed thoroughly. 1 ml aliquots were dispensedinto 1.5 ml microcentrifuge tubes, and stored at −20° C., no longer thanone year.

Annealing buffer, 10×: 10× Annealing buffer included 200 mM Tris (pH7.5) and 50 mM magnesium acetate. For this solution, 24.23 g Tris wasadded to 800 ml nH₂O and adjusted to pH 7.5. To this, 10.72 g magnesiumacetate was added and dissolved completely. The solution was brought upto a final volume of 1000 ml. The solution was able be stored at 4° C.for 1 month.

10× TE: 10× TE included 100 mM Tris HCl (pH 7.5), and 50 mM EDTA. Thesereagents were added together and mixed thoroughly. The solution could bestored at room temperature for 6 months.

Example 4 Bead Emulsion PCR

The following procedures, including capture of the template DNA, DNAamplification, and recovery of the beads bound to amplified template,can be performed in a single tube. The emulsion format ensures thephysical separation of the beads into 100-200 μm “microreactors” withinthis single tube, thus allowing for clonal amplification of the varioustemplates. Immobilization of the amplification product is achievedthrough extension of the template along the oligonucleotides bound tothe DNA capture beads. Typical, the copy number of the immobilizedtemplate ranges from 10 to 30 million copies per bead. The DNA capturebeads affixed with multiple copies of a single species of nucleic acidtemplate are ready for distribution onto PTPs.

The 300,000 75-picoliter wells etched in the PTP surface provide aunique array for the sequencing of short DNA templates in a massivelyparallel, efficient and cost-effective manner. However, this requiresfairly large quantities (millions of copies) of clonal templates in eachreaction well. The methods of the invention allow the user to clonallyamplify single-stranded genomic template species thorough PCR reactionsconducted in standard tubes or microtiter plates. Single copies of thetemplate species may be mixed with capture beads, resuspended intocomplete PCR amplification solution, and emulsified into microreactors(100 to 200 μm in diameter), after which PCR amplification generates10⁷-fold amplification of the initial template species. This procedureis much simpler and more cost-effective than previous methods.

Binding Nucleic Acid Template to Capture Beads

This example describes preparation of a population of beads thatpreferably have only one unique nucleic acid template attached thereto.Successful clonal amplification depends on the delivery of a controllednumber of template species (0.5 to 1) to each bead. Delivery of excessspecies can result in PCR amplification of a mixed template population,preventing generation of meaningful sequence data while a deficiency ofspecies will result in fewer wells containing template for sequencing.This can reduce the extent of genome coverage provided by the sequencingphase. As a result, it is preferred that the template concentration beaccurately determined through replicated quantitation, and that thebinding protocol be followed as outlined below.

Template Quality Control

The success of the Emulsion PCR reaction is related to the quality ofthe template species. Regardless of the care and detail paid to theamplification phase, poor quality templates will impede successfulamplification and the generation of meaningful sequence data. To preventunnecessary loss of time and money, it is important to check the qualityof the template material before initiating the Emulsion PCR phase of theprocess. Preferably, the library should pass two quality control stepsbefore it is used in Emulsion PCR. Its concentration and thedistribution of products it contains should be determined. Ideally, thelibrary should appear as a heterogeneous population of fragments withlittle or no visible adapter dimers (e.g., ˜90 bases). Also,amplification with PCR primers should result in a product smear ranging,for example, from 300 to 500 bp. Absence of amplification product mayreflect failure to properly ligate the adaptors to the template, whilethe presence of a single band of any size may reflect contamination ofthe template.

Preparation of the PCR Solution

The main consideration for this phase is to prevent contamination of thePCR reaction mixture with stray amplicons. Contamination of the PCRreactions with a residual amplicon is one of the critical issues thatcan cause failure of a sequencing run. To reduce the possibility ofcontamination, proper lab technique should be followed, and reactionmixture preparation should be conducted in a clean room in a UV-treatedlaminar flow hood.

PCR Reaction Mix:

For 200 μl PCR reaction mixture (enough for amplifying 600,000 beads),the following reagents were combined in a 0.2 ml PCR tube:

TABLE 4 Stock Final Microliters HIFI Buffer 10 X 1 X 20 treatednucleotides 10 mM 1 mM 20 Mg 50 mM 2 mM 8 BSA 10% 0.1% 2 Tween 80 1%0.01% 2 Ppase 2 U 0.003 U 0.333333 Primer MMP1a 100 μM 0.625 μM 1.25Primer MMP1b 10 μM 0.078 μM 1.56 Taq polymerase 5 U 0.2 U 8 Water 136.6Total 200

The tube was vortexed thoroughly and stored on ice until the beads areannealed with template.

DNA Capture Beads:

1. 600,000 DNA capture beads were transferred from the stock tube to a1.5 ml microfuge tube. The exact amount used will depend on beadconcentration of formalized reagent.

2. The beads were pelleted in a benchtop mini centrifuge and supernatantwas removed.

3. Steps 4-11 were performed in a PCR Clean Room.

4. The beads were washed with 1 mL of 1× Annealing Buffer.

5. The capture beads were pelleted in the microcentrifuge. The tube wasturned 180° and spun again.

6. All but approximately 10 μl of the supernatant was removed from thetube containing the beads. The beads were not disturbed.

7. 1 mL of 1× Annealing Buffer was added and this mixture was incubatedfor 1 minute. The beads were then pelleted as in step 5.

8. All but approximately 100 μL of the material from the tube wasremoved.

9. The remaining beads and solution were transferred to a PCR tube.

10. The 1.5 mL tube was washed with 150 μL of 1× Annealing Buffer bypipetting up and down several times. This was added to the PCR tubecontaining the beads.

11. The beads were pelleted as in step 5 and all but 10 μL ofsupernatant was removed, taking care to not disturb the bead pellet.

12. An aliquot of quantitated single-stranded template DNA (sstDNA) wasremoved. The final concentration was 200,000-sst DNA molecules/μl.

13. 3 μl of the diluted sstDNA was added to PCR tube containing thebeads. This was equivalent to 600,000 copies of sstDNA.

14. The tube was vortexed gently to mix contents.

15. The sstDNA was annealed to the capture beads in a PCR thermocyclerwith the program 80Anneal stored in the EPCR folder on the MJThermocycler, using the following protocol:

-   -   5 minutes at 65° C.;    -   Decrease by 0.1° C./sec to 60° C.;    -   Hold at 60° C. for 1 minute;    -   Decrease by 0.1° C./sec to 50° C.;    -   Hold at 50° C. for 1 minute;    -   Decrease by 0.1° C./sec to 40° C.;    -   Hold at 40° C. for 1 minute;    -   Decrease by 0.1° C./sec to 20° C.; and    -   Hold at 10° C. until ready for next step.

In most cases, beads were used for amplification immediately aftertemplate binding. If beads were not used immediately, they should werestored in the template solution at 4° C. until needed. After storage,the beads were treated as follows.

16. As in step 6, the beads were removed from the thermocycler,centrifuged, and annealing buffer was removed without disturbing thebeads.

17. The beads were stored in an ice bucket until emulsification (Example2).

18. The capture beads included, on average, 0.5 to 1 copies of sstDNAbound to each bead, and were ready for emulsification.

Example 5 Emulsification

A PCR solution suitable for use in this step is described below. For 200μl PCR reaction mix (enough for amplifying 600K beads), the followingwere added to a 0.2 ml

PCR Tube:

TABLE 4 Stock Final Microliters HIFI Buffer 10 X 1 X 20 treated Nukes 10mM 1 mM 20 Mg 50 mM 2 mM 8 BSA 10% 0.1% 2 Tween 80 1% 0.01% 2 Ppase 2 U0.003 U 0.333333 Primer MMP1a 100 μM 0.625 μM 1.25 Primer MMP1b 10 μM0.078 μM 1.56 Taq 5 U 0.2 U 8 Water 136.6 Total 200

This example describes how to create a heat-stable water-in-oil emulsioncontaining about 3,000 PCR microreactors per microliter. Outlined belowis a protocol for preparing the emulsion.

1. 200 μl of PCR solution was added to the 600,000 beads (bothcomponents from Example 1).

2. The solution was pipetted up and down several times to resuspend thebeads.

3. The PCR-bead mixture was allowed to incubate at room temperature for2 minutes to equilibrate the beads with PCR solution.

4. 400 μl of Emulsion Oil was added to a UV-irradiated 2 ml microfugetube.

5. An “amplicon-free” ¼″ stir magnetic stir bar was added to the tube ofEmulsion Oil.

An amplicon-free stir bar was prepared as follows. A large stir bar wasused to hold a ¼″ stir bar. The stir bar was then:

-   -   Washed with DNA-Off (drip or spray);    -   Rinsed with picopure water;    -   Dried with a Kimwipe edge; and    -   UV irradiated for 5 minutes.

6. The magnetic insert of a Dynal MPC-S tube holder was removed. Thetube of Emulsion Oil was placed in the tube holder. The tube was set inthe center of a stir plate set at 600 rpm.

7. The tube was vortexed extensively to resuspend the beads. Thisensured that there was minimal clumping of beads.

8. Using a P-200 pipette, the PCR-bead mixture was added drop-wise tothe spinning oil at a rate of about one drop every 2 seconds, allowingeach drop to sink to the level of the magnetic stir bar and becomeemulsified before adding the next drop. The solution turned into ahomogeneous milky white liquid with a viscosity similar to mayonnaise.

9. Once the entire PCR-bead mixture was been added, the microfuge tubewas flicked a few times to mix any oil at the surface with the milkyemulsion.

10. Stirring was continued for another 5 minutes.

11. Steps 9 and 10 were repeated.

12. The stir bar was removed from the emulsified material by dragging itout of the tube with a larger stir bar.

13. 10 μL of the emulsion was removed and placed on a microscope slide.The emulsion was covered with a cover slip and the emulsion wasinspected at 50× magnification (10× ocular and 5× objective lens). A“good” emulsion was expected to include primarily single beads inisolated droplets (microreactors) of PCR solution in oil.

14. A suitable emulsion oil mixture with emulsion stabilizers was madeas follows. The components for the emulsion mixture are shown in Table5.

TABLE 5 Quantity Ingredient Required Source Ref. Number Sigma LightMineral Oil 94.5 g Sigma M-5904 Atlox 4912   1 g Uniqema NA Span 80  4.5g Uniqema NA

The emulsion oil mixture was made by prewarming the Atlox 4912 to 60° C.in a water bath. Then, 4.5 grams of Span 80 was added to 94.5 grams ofmineral oil to form a mixture. Then, one gram of the prewarmed Atlox4912 was added to the mixture. The solutions were placed in a closedcontainer and mixed by shaking and inversion. Any sign that the Atloxwas settling or solidifying was remedied by warming the mixture to 60°C., followed by additional shaking.

Example 6 Amplification

This example describes amplification of the template DNA in thebead—emulsion mixture. According to this protocol of the invention, theDNA amplification phase of the process takes 3 to 4 hours. After theamplification is complete, the emulsion may be left on the thermocyclerfor up to 12 hours before beginning the process of isolating the beads.PCR thermocycling was performed by placing 50 to 100 μl of theemulsified reaction mixture into individual PCR reaction chambers (i.e.,PCR tubes). PCR was performed as follows:

1. The emulsion was transferred in 50-100 μL amounts into approximately10 separate PCR tubes or a 96-well plate using a single pipette tip. Forthis step, the water-in-oil emulsion was highly viscous.

2. The plate was sealed, or the PCR tube lids were closed, and thecontainers were placed into in a MJ thermocycler with or without a96-well plate adaptor.

3. The PCR thermocycler was programmed to run the following program:

-   -   1 cycle (4 minutes at 94° C.)—Hotstart Initiation;    -   40 cycles (30 seconds at 94° C., 30 seconds at 58° C., 90        seconds at 68° C.);    -   25 cycles (30 seconds at 94° C., 6 minutes at 58° C.); and    -   Storage at 14° C.

4. After completion of the PCR reaction, the amplified material wasremoved in order to proceed with breaking the emulsion and beadrecovery.

Example 7 Breaking the Emulsion and Bead Recovery

This example describes how to break the emulsion and recover the beadswith amplified template thereon. Preferably, the post-PCR emulsionshould remain intact. The lower phase of the emulsion should, by visualinspection, remain a milky white suspension. If the solution is clear,the emulsion may have partially resolved into its aqueous and oilphases, and it is likely that many of the beads will have a mixture oftemplates. If the emulsion has broken in one or two of the tubes, thesesamples should not be combined with the others. If the emulsion hasbroken in all of the tubes, the procedure should not be continued.

1. All PCR reactions from the original 600 μl sample were combined intoa single 1.5 ml microfuge tube using a single pipette tip. As indicatedabove, the emulsion was quite viscous. In some cases, pipetting wasrepeated several times for each tube. As much material as possible wastransferred to the 1.5 ml tube.

2. The remaining emulsified material was recovered from each PCR tube byadding 50 μl of Sigma Mineral Oil into each sample. Using a singlepipette tip, each tube was pipetted up and down a few times to resuspendthe remaining material.

3. This material was added to the 1.5 ml tube containing the bulk of theemulsified material.

4. The sample was vortexed for 30 seconds.

5. The sample was spun for 20 minutes in the tabletop microfuge tube at13.2K rpm in the Eppendorf microcentrifuge.

6. The emulsion separated into two phases with a large white interface.As much of the top, clear oil phase as possible was removed. The cloudymaterial was left in the tube. Often a white layer separated the oil andaqueous layers. Beads were often observed pelleted at the bottom of thetube.

7. The aqueous layer above the beads was removed and saved for analysis(gel analysis, Agilent 2100, and Taqman). If an interface of whitematerial persisted above the aqueous layer, 20 microliters of theunderlying aqueous layer was removed. This was performed by penetratingthe interface material with a pipette tip and withdrawing the solutionfrom underneath.

8. In the PTP Fabrication and Surface Chemistry Room Fume Hood, 1 ml ofHexanes was added to the remainder of the emulsion.

9. The sample was vortexed for 1 minute and spun at full speed for 1minute.

10. In the PTP Fabrication and Surface Chemistry Room Fume Hood, thetop, oil/hexane phase was removed and placed into the organic wastecontainer.

11. 1 ml of 1× Annealing Buffer was added in 80% Ethanol to theremaining aqueous phase, interface, and beads.

12. The sample was vortexed for 1 minute or until the white substancedissolved.

13. The sample was centrifuged for 1 minute at high speed. The tube wasrotated 180 degrees, and spun again for 1 minute. The supernatant wasremoved without disturbing the bead pellet.

14. The beads were washed with 1 ml of 1× Annealing Buffer containing0.1% Tween 20 and this step was repeated.

Example 8 Single Strand Removal and Primer Annealing

If the beads are to be used in a pyrophosphate-based sequencingreaction, then it is necessary to remove the second strand of the PCRproduct and anneal a sequencing primer to the single stranded templatethat is bound to the bead. This example describes a protocol foraccomplishing that.

1. The beads were washed with 1 ml of water, and spun twice for 1minute. The tube was rotated 180° between spins. After spinning, theaqueous phase was removed.

2. The beads were washed with 1 ml of 1 mM EDTA. The tube was spun as instep 1 and the aqueous phase was removed.

3. 1 ml of 0.125 M NaOH was added and the sample was incubated for 8minutes.

4. The sample was vortexed briefly and placed in a microcentrifuge.

5. After 6 minutes, the beads were pelleted as in step 1 and as muchsolution as possible was removed.

6. At the completion of the 8 minute NaOH incubation, 1 ml of 1×Annealing Buffer was added.

7. The sample was briefly vortexed, and the beads were pelleted as instep 1. As much supernatant as possible was removed, and another 1 ml of1× Annealing buffer was added.

8. The sample was briefly vortexed, the beads were pelleted as in step1, and 800 μl of 1× Annealing Buffer was removed.

9. The beads were transferred to a 0.2 ml PCR tube.

10. The beads were transferred and as much Annealing Buffer as possiblewas removed, without disturbing the beads.

11. 100 μl of 1× Annealing Buffer was added.

12. 4 μl of 100 μM sequencing primer was added. The sample was vortexedjust prior to annealing.

13. Annealing was performed in a MJ thermocycler using the “80Anneal”program.

14. The beads were washed three times with 200 μl of 1× Annealing Bufferand resuspended with 100 μl of 1× Annealing Buffer.

15. The beads were counted in a Hausser Hemacytometer. Typically,300,000 to 500,000 beads were recovered (3,000-5,000 beads/μL).

16. Beads were stored at 4° C. and could be used for sequencing for 1week.

Example 9 Optional Enrichment Step

The beads may be enriched for amplicon containing bead using thefollowing procedure. Enrichment is not necessary but it could be used tomake subsequent molecular biology techniques, such as DNA sequencing,more efficient.

Fifty microliters of 10 μM (total 500 pmoles) of biotin-sequencingprimer was added to the Sepharose beads containing amplicons fromExample 5. The beads were placed in a thermocycler. The primer wasannealed to the DNA on the bead by the thermocycler annealing program ofExample 2.

After annealing, the sepharose beads were washed three times withAnnealing Buffer containing 0.1% Tween 20. The beads, now containingssDNA fragments annealed with biotin-sequencing primers, wereconcentrated by centrifugation and resuspended in 200 μl of BST bindingbuffer. Ten microliters of 50,000 unit/ml Bst-polymerase was added tothe resuspended beads and the vessel holding the beads was placed on arotator for five minutes. Two microliters of 10 mM dNTP mixture (i.e.,2.5 μl each of 10 mM dATP, dGTP, dCTP and dTTP) was added and themixture was incubated for an additional 10 minutes at room temperature.The beads were washed three times with annealing buffer containing 0.1%Tween 20 and resuspended in the original volume of annealing buffer.

Fifty microliters of Dynal Streptavidin beads (Dynal Biotech Inc., LakeSuccess, N.Y.; M270 or MyOne™ beads at 10 mg/ml) was washed three timeswith Annealing Buffer containing 0.1% Tween 20 and resuspended in theoriginal volume in Annealing Buffer containing 0.1% Tween 20. Then theDynal bead mixture was added to the resuspended sepharose beads. Themixture was vortexed and placed in a rotator for 10 minutes at roomtemperature.

The beads were collected on the bottom of the test tube bycentrifugation at 2300 g (500 rpm for Eppendorf Centrifuge 5415D). Thebeads were resuspended in the original volume of Annealing Buffercontaining 0.1% Tween 20. The mixture, in a test tube, was placed in amagnetic separator (Dynal). The beads were washed three times withAnnealing Buffer containing 0.1% Tween 20 and resuspended in theoriginal volume in the same buffer. The beads without amplicons wereremoved by wash steps, as previously described. Only Sepharose beadscontaining the appropriated DNA fragments were retained.

The magnetic beads were separated from the sepharose beads by additionof 500 μl of 0.125 M NaOH. The mixture was vortexed and the magneticbeads were removed by magnetic separation. The Sepharose beads remainingin solution was transferred to another tube and washed with 400 μl of 50mM Tris Acetate until the pH was stabilized at 7.6.

Example 10 Nucleic Acid Sequencing Using Bead Emulsion PCR

The following experiment was performed to test the efficacy of the beademulsion PCR. For this protocol, 600,000 Sepharose beads, with anaverage diameter of 25-35 μm (as supplied my the manufacturer) werecovalently attached to capture primers at a ratio of 30-50 millioncopies per bead. The beads with covalently attached capture primers weremixed with 1.2 million copies of single stranded Adenovirus Library. Thelibrary constructs included a sequence that was complimentary to thecapture primer on the beads.

The adenovirus library was annealed to the beads using the proceduredescribed in Example 1. Then, the beads were resuspended in complete PCRsolution. The PCR Solution and beads were emulsified in 2 volumes ofspinning emulsification oil using the same procedure described inExample 2. The emulsified (encapsulated) beads were subjected toamplification by PCR as outlined in Example 3. The emulsion was brokenas outlined in Example 4. DNA on beads was rendered single stranded,sequencing primer was annealed using the procedure of Example 5.

Next, 70,000 beads were sequenced simultaneously by pyrophosphatesequencing using a pyrophosphate sequencer from 454 Life Sciences (NewHaven, Conn.) (see co-pending application of Lohman et al., filedconcurrently herewith entitled Methods of Amplifying and SequencingNucleic Acids” U.S. Ser. No. 60/476,592 filed Jun. 6, 2003). Multiplebatches of 70,000 beads were sequenced and the data were listed in Table6, below.

TABLE 6 Alignment Inferred Error Alignments Read Tolerance None SingleMultiple Unique Coverage Error 0% 47916 1560 1110 54.98% 0.00% 5% 460263450 2357 83.16% 1.88% 10%  43474 6001 1 3742 95.64% 4.36%

This table shows the results obtained from BLAST analysis comparing thesequences obtained from the pyrophosphate sequencer against Adenovirussequence. The first column shows the error tolerance used in the BLASTprogram. The last column shows the real error as determined by directcomparison to the known sequence.

Bead Emulsion PCR for Double Ended Sequencing

Example 11 Template Quality Control

As indicated previously, the success of the Emulsion PCR reaction wasfound to be related to the quality of the single stranded templatespecies. Accordingly, the quality of the template material was assessedwith two separate quality controls before initiating the Emulsion PCRprotocol. First, an aliquot of the single-stranded template was run onthe 2100 BioAnalyzer (Agilient). An RNA Pico Chip was used to verifythat the sample included a heterogeneous population of fragments,ranging in size from approximately 200 to 500 bases. Second, the librarywas quantitated using the RiboGreen fluorescence assay on a Bio-TekFL600 plate fluorometer. Samples determined to have DNA concentrationsbelow 5 ng/μl were deemed too dilute for use.

Example 12 DNA Capture Bead Synthesis

Packed beads from a 1 mL N-hydroxysuccinimide ester (NHS)-activatedSepharose HP affinity column (Amersham Biosciences, Piscataway, N.J.)were removed from the column. The 30-25 μm size beads were selected byserial passage through 30 and 25 μm pore filter mesh sections (SefarAmerica, Depew, N.Y., USA). Beads that passed through the first filter,but were retained by the second were collected and activated asdescribed in the product literature (Amersham Pharmacia Protocol #71700600AP). Two different amine-labeled HEG (hexaethyleneglycol) longcapture primers were obtained, corresponding to the 5′ end of the senseand antisense strand of the template to be amplified, (5′-Amine-3 HEGspacers gcttacctgaccgacctctgcctatcccctgttgcgtgtc-3′; SEQ ID NO: 12; and5′-Amine-3 HEG spacers ccattccccagctcgtcttgccatctgttccctccctgtc-3′; SEQID NO: 13) (IDT Technologies, Coralville, Iowa, USA). The primers weredesigned to capture of both strands of the amplification products toallow double ended sequencing, i.e., sequencing the first and secondstrands of the amplification products. The capture primers weredissolved in 20 mM phosphate buffer, pH 8.0, to obtain a finalconcentration of 1 mM. Three microliters of each primer were bound tothe sieved 30-25 μm beads. The beads were then stored in a bead storagebuffer (50 mM Tris, 0.02% Tween and 0.02% sodium azide, pH 8). The beadswere quantitated with a hemacytometer (Hausser Scientific, Horsham, Pa.,USA) and stored at 4° C. until needed.

Example 13 PCR Reaction Mix Preparation and Formulation

As with any single molecule amplification technique, contamination ofthe reactions with foreign or residual amplicon from other experimentscould interfere with a sequencing run. To reduce the possibility ofcontamination, the PCR reaction mix was prepared in a in a UV-treatedlaminar flow hood located in a PCR clean room. For each 600,000 beademulsion PCR reaction, the following reagents were mixed in a 1.5 mltube: 225 μl of reaction mixture (1× Platinum HiFi Buffer (Invitrogen)),1 mM dNTPs, 2.5 mM MgSO₄ (Invitrogen), 0.1% BSA, 0.01% Tween, 0.003 U/μlthermostable PPi-ase (NEB), 0.125 μM forward primer(5′-gcttacctgaccgacctctg-3′; SEQ ID NO:14) and 0.125 μM reverse primer(5′-ccattccccagctcgtcttg-3′; SEQ ID NO:15) (IDT Technologies,Coralville, Iowa, USA) and 0.2 U/μl Platinum Hi-Fi Taq Polymerase(Invitrogen). Twenty-five microliters of the reaction mixture wasremoved and stored in an individual 200 μl PCR tube for use as anegative control. Both the reaction mixture and negative controls werestored on ice until needed.

Example 14 Binding Template Species to DNA Capture Beads

Successful clonal DNA amplification for sequencing relates to thedelivery of a controlled number of template species to each bead. Forthe experiments described herein below, the typical target templateconcentration was determined to be 0.5 template copies per capture bead.At this concentration, Poisson distribution dictates that 61% of thebeads have no associated template, 30% have one species of template, and9% have two or more template species. Delivery of excess species canresult in the binding and subsequent amplification of a mixed population(2 or more species) on a single bead, preventing the generation ofmeaningful sequence data. However, delivery of too few species willresult in fewer wells containing template (one species per bead),reducing the extent of sequencing coverage. Consequently, it was deemedthat the single-stranded library template concentration was important.

Template nucleic acid molecules were annealed to complimentary primerson the DNA capture beads by the following method, conducted in aUV-treated laminar flow hood. Six hundred thousand DNA capture beadssuspended in bead storage buffer (see Example 9, above) were transferredto a 200 μl PCR tube. The tube was centrifuged in a benchtop minicentrifuge for 10 seconds, rotated 180°, and spun for an additional 10seconds to ensure even pellet formation. The supernatant was removed,and the beads were washed with 200 μl of Annealing Buffer (20 mM Tris,pH 7.5 and 5 mM magnesium acetate). The tube was vortexed for 5 secondsto resuspend the beads, and the beads were pelleted as before. All butapproximately 10 μl of the supernatant above the beads was removed, andan additional 200 μl of Annealing Buffer was added. The beads were againvortexed for 5 seconds, allowed to sit for 1 minute, and then pelletedas before. All but 10 μl of supernatant was discarded.

Next, 1.5 μl of 300,000 molecules/μl template library was added to thebeads. The tube was vortexed for 5 seconds to mix the contents, and thetemplates were annealed to the beads in a controlleddenaturation/annealing program preformed in an MJ thermocycler. Theprogram allowed incubation for 5 minutes at 80° C., followed by adecrease by 0.1° C./sec to 70° C., incubation for 1 minute at 70° C.,decrease by 0.1° C./sec to 60° C., hold at 60° C. for 1 minute, decreaseby 0.1° C./sec to 50° C., hold at 50° C. for 1 minute, decrease by 0.1°C./sec to 20° C., hold at 20° C. Following completion of the annealingprocess, the beads were removed from the thermocycler, centrifuged asbefore, and the Annealing Buffer was carefully decanted. The capturebeads included on average 0.5 copy of single stranded template DNA boundto each bead, and were stored on ice until needed.

Example 15 Emulsification

The emulsification process creates a heat-stable water-in-oil emulsioncontaining 10,000 discrete PCR microreactors per microliter. This servesas a matrix for single molecule, clonal amplification of the individualmolecules of the target library. The reaction mixture and DNA capturebeads for a single reaction were emulsified in the following manner. Ina UV-treated laminar flow hood, 200 μl of PCR solution (from Example 10)was added to the tube containing the 600,000 DNA capture beads (fromExample 11). The beads were resuspended through repeated pipetting.After this, the PCR-bead mixture was incubated at room temperature forat least 2 minutes, allowing the beads to equilibrate with the PCRsolution. At the same time, 450 μl of Emulsion Oil (4.5% (w:w) Span 80,1% (w:w) Atlox 4912 (Uniqema, Del.) in light mineral oil (Sigma)) wasaliquotted into a flat-topped 2 ml centrifuge tube (Dot Scientific)containing a sterile ¼ inch magnetic stir bar (Fischer). This tube wasthen placed in a custom-made plastic tube holding jig, which was thencentered on a Fisher Isotemp digital stirring hotplate (FisherScientific) set to 450 RPM.

The PCR-bead solution was vortexed for 15 seconds to resuspend thebeads. The solution was then drawn into a 1 ml disposable plasticsyringe (Benton-Dickenson) affixed with a plastic safety syringe needle(Henry Schein). The syringe was placed into a syringe pump (Cole-Parmer)modified with an aluminum base unit orienting the pump vertically ratherthan horizontally (FIG. 30). The tube with the emulsion oil was alignedon the stir plate so that it was centered below the plastic syringeneedle and the magnetic stir bar was spinning properly. The syringe pumpwas set to dispense 0.6 ml at 5.5 ml/hr. The PCR-bead solution was addedto the emulsion oil in a dropwise fashion. Care was taken to ensure thatthe droplets did not contact the side of the tube as they fell into thespinning oil.

Once the emulsion was formed, great care was taken to minimize agitationof the emulsion during both the emulsification process and thepost-emulsification aliquotting steps. It was found that vortexing,rapid pipetting, or excessive mixing could cause the emulsion to break,destroying the discrete microreactors. In forming the emulsion, the twosolutions turned into a homogeneous milky white mixture with theviscosity of mayonnaise. The contents of the syringe were emptied intothe spinning oil. Then, the emulsion tube was removed from the holdingjig, and gently flicked with a forefinger until any residual oil layerat the top of the emulsion disappeared. The tube was replaced in theholding jig, and stirred with the magnetic stir bar for an additionalminute. The stir bar was removed from the emulsion by running a magneticretrieval tool along the outside of the tube, and the stir bar wasdiscarded.

Twenty microliters of the emulsion was taken from the middle of the tubeusing a P100 pipettor and placed on a microscope slide. The largerpipette tips were used to minimize shear forces. The emulsion wasinspected at 50× magnification to ensure that it was comprisedpredominantly of single beads in 30 to 150 micron diameter microreactorsof PCR solution in oil (FIG. 33). After visual examination, theemulsions were immediately amplified.

Example 16 Amplification

The emulsion was aliquotted into 7-8 separate PCR tubes. Each tubeincluded approximately 75 μl of the emulsion. The tubes were sealed andplaced in a MJ thermocycler along with the 25 μl negative controldescribed above. The following cycle times were used: 1 cycle ofincubation for 4 minutes at 94° C. (Hotstart Initiation), 30 cycles ofincubation for 30 seconds at 94° C., and 150 seconds at 68° C.(Amplification), and 40 cycles of incubation for 30 seconds at 94° C.,and 360 seconds at 68° C. (Hybridization and Extension). Aftercompletion of the PCR program, the tubes were removed and the emulsionswere broken immediately or the reactions were stored at 10° C. for up to16 hours prior to initiating the breaking process.

Example 17 Breaking the Emulsion and Bead Recovery

Following amplification, the emulstifications were examined for breakage(separation of the oil and water phases). Unbroken emulsions werecombined into a single 1.5 ml microcentrifuge tube, while the occasionalbroken emulsion was discarded. As the emulsion samples were quiteviscous, significant amounts remained in each PCR tube. The emulsionremaining in the tubes was recovered by adding 75 μl of mineral oil intoeach PCR tube and pipetting the mixture. This mixture was added to the1.5 ml tube containing the bulk of the emulsified material. The 1.5 mltube was then vortexed for 30 seconds. After this, the tube wascentrifuged for 20 minutes in the benchtop microcentrifuge at 13.2K rpm(full speed).

After centrifugation, the emulsion separated into two phases with alarge white interface. The clear, upper oil phase was discarded, whilethe cloudy interface material was left in the tube. In a chemical fumehood, 1 ml hexanes was added to the lower phase and interface layer. Themixture was vortexed for 1 minute and centrifuged at full speed for 1minute in a benchtop microcentrifuge. The top, oil/hexane phase wasremoved and discarded. After this, 1 ml of 80% Ethanol/1× AnnealingBuffer was added to the remaining aqueous phase, interface, and beads.This mixture was vortexed for 1 minute or until the white material fromthe interface was dissolved. The sample was then centrifuged in abenchtop microcentrifuge for 1 minute at full speed. The tube wasrotated 180 degrees, and spun again for an additional minute. Thesupernatant was then carefully removed without disturbing the beadpellet.

The white bead pellet was washed twice with 1 ml Annealing Buffercontaining 0.1% Tween 20. The wash solution was discarded and the beadswere pelleted after each wash as described above. The pellet was washedwith 1 ml Picopure water. The beads were pelleted with thecentrifuge-rotate-centrifuge method used previously. The aqueous phasewas carefully removed. The beads were then washed with 1 ml of 1 mM EDTAas before, except that the beads were briefly vortexed at a mediumsetting for 2 seconds prior to pelleting and supernatant removal.

Amplified DNA, immobilized on the capture beads, was treated to obtainsingle stranded DNA. The second strand was removed by incubation in abasic melt solution. One ml of Melt Solution (0.125 M NaOH, 0.2 M NaCl)was subsequently added to the beads. The pellet was resuspended byvortexing at a medium setting for 2 seconds, and the tube placed in aThermolyne LabQuake tube roller for 3 minutes. The beads were thenpelleted as above, and the supernatant was carefully removed anddiscarded. The residual Melt solution was neutralized by the addition of1 ml Annealing Buffer. After this, the beads were vortexed at mediumspeed for 2 seconds. The beads were pelleted, and the supernatant wasremoved as before. The Annealing Buffer wash was repeated, except thatonly 800 μl of the Annealing Buffer was removed after centrifugation.The beads and remaining Annealing Buffer were transferred to a 0.2 mlPCR tube. The beads were used immediately or stored at 4° C. for up to48 hours before continuing on to the enrichment process.

Example 18 Optional Bead Enrichment

The bead mass included beads with amplified, immobilized DNA strands,and empty or null beads. As mentioned previously, it was calculated that61% of the beads lacked template DNA during the amplification process.Enrichment was used to selectively isolate beads with template DNA,thereby maximizing sequencing efficiency. The enrichment process isdescribed in detail below.

The single stranded beads from Example 14 were pelleted with thecentrifuge-rotate-centrifuge method, and as much supernatant as possiblewas removed without disturbing the beads. Fifteen microliters ofAnnealing Buffer were added to the beads, followed by 2 μl of 100 μMbiotinylated, 40 base enrichment primer (5′-Biotin-tetra-ethyleneglycolspacers ccattccccagctcgtcttgccatctgttccctccctgtctcag-3′; SEQ ID NO:16).The primer was complimentary to the combined amplification andsequencing sites (each 20 bases in length) on the 3′ end of thebead-immobilized template. The solution was mixed by vortexing at amedium setting for 2 seconds, and the enrichment primers were annealedto the immobilized DNA strands using a controlled denaturation/annealingprogram in an MJ thermocycler. The program consisted of the followingcycle times and temperatures: incubation for 30 seconds at 65° C.,decrease by 0.1° C./sec to 58° C., incubation for 90 seconds at 58° C.,and hold at 10° C.

While the primers were annealing, Dynal MyOne™ streptavidin beads wereresuspend by gentle swirling. Next, 20 μl of the MyOne™ beads were addedto a 1.5 ml microcentrifuge tube containing 1 ml of Enhancing fluid (2 MNaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5). The MyOne bead mixture wasvortexed for 5 seconds, and the tube was placed in a Dynal MPC-S magnet.The paramagnetic beads were pelleted against the side of themicrocentrifuge tube. The supernatant was carefully removed anddiscarded without disturbing the MyOne™ beads. The tube was removed fromthe magnet, and 100 μl of enhancing fluid was added. The tube wasvortexed for 3 seconds to resuspend the beads, and stored on ice untilneeded.

Upon completion of the annealing program, 100 μl of annealing buffer wasadded to the PCR tube containing the DNA capture beads and enrichmentprimer. The tube vortexed for 5 seconds, and the contents weretransferred to a fresh 1.5 ml microcentrifuge tube. The PCR tube inwhich the enrichment primer was annealed to the capture beads was washedonce with 200 μl of annealing buffer, and the wash solution was added tothe 1.5 ml tube. The beads were washed three times with 1 ml ofannealing buffer, vortexed for 2 seconds, and pelleted as before. Thesupernatant was carefully removed. After the third wash, the beads werewashed twice with 1 ml of ice cold Enhancing fluid. The beads werevortexed, pelleted, and the supernatant was removed as before. The beadswere resuspended in 150 μl ice cold Enhancing fluid and the beadsolution was added to the washed MyOne™ beads.

The bead mixture was vortexed for 3 seconds and incubated at roomtemperature for 3 minutes on a LabQuake tube roller. Thestreptavidin-coated MyOne™ beads were bound to the biotinylatedenrichment primers annealed to immobilized templates on the DNA capturebeads. The beads were then centrifuged at 2,000 RPM for 3 minutes, afterwhich the beads were vortexed with 2 second pulses until resuspended.The resuspended beads were placed on ice for 5 minutes. Following this,500 μl of cold Enhancing fluid was added to the beads and the tube wasinserted into a Dynal MPC-S magnet. The beads were left undisturbed for60 seconds to allow pelleting against the magnet. After this, thesupernatant with excess MyOne™ and null DNA capture beads was carefullyremoved and discarded.

The tube was removed from the MPC-S magnet, and 1 ml of cold enhancingfluid added to the beads. The beads were resuspended with gentle fingerflicking. It was important not to vortex the beads at this time, asforceful mixing could break the link between the MyOne™ and DNA capturebeads. The beads were returned to the magnet, and the supernatantremoved. This wash was repeated three additional times to ensure removalof all null capture beads. To remove the annealed enrichment primers andMyOne™ beads, the DNA capture beads were resuspended in 400 μl ofmelting solution, vortexed for 5 seconds, and pelleted with the magnet.The supernatant with the enriched beads was transferred to a separate1.5 ml microcentrifuge tube. For maximum recovery of the enriched beads,a second 400 μl aliquot of melting solution was added to the tubecontaining the MyOne™ beads. The beads were vortexed and pelleted asbefore. The supernatant from the second wash was removed and combinedwith the first bolus of enriched beads. The tube of spent MyOne™ beadswas discarded.

The microcentrifuge tube of enriched DNA capture beads was placed on theDynal MPC-S magnet to pellet any residual MyOne™ beads. The enrichedbeads in the supernatant were transferred to a second 1.5 mlmicrocentrifuge tube and centrifuged. The supernatant was removed, andthe beads were washed 3 times with 1 ml of annealing buffer toneutralize the residual melting solution. After the third wash, 800 μlof the supernatant was removed, and the remaining beads and solutionwere transferred to a 0.2 ml PCR tube. The enriched beads werecentrifuged at 2,000 RPM for 3 minutes and the supernatant decanted.Next, 20 μl of annealing buffer and 3 μl of two different 100 μMsequencing primers (5′-ccatctgttccctccctgtc-3′; SEQ ID NO:17; and5′-cctatcccctgttgcgtgtc-3′ phosphate; SEQ ID NO:18) were added. The tubewas vortexed for 5 seconds, and placed in an MJ thermocycler for thefollowing 4-stage annealing program: incubation for 5 minutes at 65° C.,decrease by 0.1° C./sec to 50° C., incubation for 1 minute at 50° C.,decrease by 0.1° C./sec to 40° C., hold at 40° C. for 1 minute, decreaseby 0.1° C./sec to 15° C., and hold at 15° C.

Upon completion of the annealing program, the beads were removed fromthermocycler and pelleted by centrifugation for 10 seconds. The tube wasrotated 180°, and spun for an additional 10 seconds. The supernatant wasdecanted and discarded, and 200 μl of annealing buffer was added to thetube. The beads were resuspended with a 5 second vortex, and pelleted asbefore. The supernatant was removed, and the beads resuspended in 100 μlannealing buffer. At this point, the beads were quantitated with aMultisizer 3 Coulter Counter (Beckman Coulter). Beads were stored at 4°C. and were stable for at least 1 week.

Example 19 Double Strand Sequencing

For double strand sequencing, two different sequencing primers are used;an unmodified primer MMP7A and a 3′ phosphorylated primer MMP2 Bp. Thereare multiple steps in the process. This process is shown schematicallyin FIG. 38.

1. First Strand Sequencing. Sequencing of the first strand involvesextension of the unmodified primer by a DNA polymerase throughsequential addition of nucleotides for a predetermined number of cycles.

2. CAPPING: The first strand sequencing was terminated by flowing aCapping Buffer containing 25 mM Tricine, 5 mM Magnesium acetate, 1 mMDTT, 0.4 mg/ml PVP, 0.1 mg/ml BSA, 0.01% Tween and 2 μM of eachdideoxynucleotides and 2 μM of each deoxynucleotide.

3. CLEAN: The residual deoxynucleotides and dideoxynucleotides wasremoved by flowing in Apyrase Buffer containing 25 mM Tricine, 5 mMMagnesium acetate, 1 mM DTT, 0.4 mg/ml PVP, 0.1 mg/ml BSA, 0.01% Tweenand 8.5 units/L of Apyrase.

4. CUTTING: The second blocked primer was unblocked by removing thephosphate group from the 3′ end of the modified 3′ phosphorylated primerby flowing a Cutting buffer containing 5 units/ml of Calf intestinalphosphatases.

5. CONTINUE: The second unblocked primer was activated by addition ofpolymerase by flowing 1000 units/ml of DNA polymerases to capture allthe available primer sites.

6. Second Strand Sequencing: Sequencing of the second strand by a DNApolymerase through sequential addition of nucleotides for apredetermined number of cycles.

Using the methods described above, the genomic DNA of Staphylococcusaureus was sequenced. The results are presented in FIG. 39. A total of31,785 reads were obtained based on 15770 reads of the first strand and16015 reads of the second strand. Of these, a total of 11,799 reads werepaired and 8187 reads were unpaired obtaining a total coverage of 38%.

Read lengths ranged from 60 to 130 with an average of 95+/−9 bases (FIG.40). The distribution of genome span and the number of wells of eachgenome span is shown in FIG. 41. Representative alignment strings, fromthis genomic sequencing, are shown in FIG. 42.

Example 20 Template PCR

30 micron NHS Sepharose beads were coupled with 1 mM of each of thefollowing primers:

MMP1A: cgtttcccctgtgtgccttg (SEQ ID NO:19)

MMP1B: ccatctgttgcgtgcgtgtc (SEQ ID NO:20)

Drive-to-bead PCR was performed in a tube on the MJ thermocycler byadding 50 μl of washed primer-coupled beads to a PCR master mix at aone-to-one volume-to-volume ratio. The PCR master mixture included:

1×PCR buffer;

1 mM of each dNTP;

0.625 μM primer MMP1A;

0.625 μM primer MMP1B;

1 μl of 1 unit/μl Hi Fi Taq (Invitrogen, San Diego, Calif.); and

˜5-10 ng Template DNA (the DNA to be sequenced).

The PCR reaction was performed by programming the MJ thermocycler forthe following: incubation at 94° C. for 3 minutes; 39 cycles ofincubation at 94° C. for 30 seconds, 58° C. for 30 seconds, 68° C. for30 seconds; followed by incubation at 94° C. for 30 seconds and 58° C.for 10 minutes; 10 cycles of incubation at 94° C. for 30 seconds, 58° C.for 30 seconds, 68° C. for 30 seconds; and storage at 10° C.

Example 21 Template DNA Preparation and Annealing Sequencing Primer

The beads from Example 1 were washed two times with distilled water;washed once with 1 mM EDTA, and incubated with 0.125 M NaOH for 5minutes. This removed the DNA strands not linked to the beads. Then, thebeads were washed once with 50 mM Tris Acetate buffer, and twice withAnnealing Buffer: 200 mM Tris-Acetate, 50 mM Mg Acetate, pH 7.5. Next,500 pmoles of Sequencing Primer MMP7A (ccatctgttccctccctgtc; SEQ IDNO:21) and MMP2B-phos (cctatcccctgttgcgtgtc; SEQ ID NO:22) were added tothe beads. The primers were annealed with the following program on theMJ thermocycler: incubation at 60° C. for 5 minutes; temperature drop of0.1 degree per second to 50° C.; incubation at 50° C. for 5 minutes;temperature drop of 0.1 degree per second to 4° C.; incubation at 40° C.for 5 minutes; temperature drop of 0.1 degree per second to 10° C. Thetemplate was then sequenced using standard pyrophosphate sequencing.

Example 22 Sequencing and Stopping of the First Strand

The beads were spun into a 55 μm PicoTiter plate (PTP) at 3000 rpm for10 minutes. The PTP was placed on a rig and run using de novo sequencingfor a predetermined number of cycles. The sequencing was stopped bycapping the first strand. The first strand was capped by adding 100 μlof 1× AB (50 mM Mg Acetate, 250 mM Tricine), 1000 unit/ml BSTpolymerase, 0.4 mg/ml single strand DNA binding protein, 1 mM DTT, 0.4mg/ml PVP (Polyvinyl Pyrolidone), 10 uM of each ddNTP, and 2.5 μM ofeach dNTP. Apyrase was then flowed over in order to remove excessnucleotides by adding 1× AB, 0.4 mg/ml PVP, 1 mM DTT, 0.1 mg/ml BSA,0.125 units/ml apyrase, incubated for 20 minutes.

Example 23 Preparation of Second Strand for Sequencing

The second strand was unblocked by adding 100 μl of 1× AB, 0.1 unit perml poly nucleotide kinase, 5 mM DTT. The resultant template wassequenced using standard pyrophosphate sequencing (described, e.g., inU.S. Pat. Nos. 6,274,320, 6,258,568 and 6,210,891, incorporated hereinby reference). The results of the sequencing method can be seen in FIG.10F where a fragment of 174 bp was sequenced on both ends usingpyrophosphate sequencing and the methods described in these examples.

Example 24 Sequence Analysis of Nucleic Acid on a Picotiter Plate

The picotiter plate containing amplified nucleic acids as described inExample 2 is placed in a perfusion chamber. Then sulfurylase, apyrase,and luciferase are delivered to the picotiter plate.

The sequencing primer primes DNA synthesis extending into the insertsuspected of having a polymorphism, as shown in FIGS. 11A-11D. Thesequencing primer is first extended by delivering into the perfusionchamber, in succession, a wash solution, a DNA polymerase, and one ofdTTP, dGTP, dCTP, or α thio dATP (a dATP analog). The sulfurylase,luciferase, and apyrase, attached to the termini convert any PPiliberated as part of the sequencing reaction to detectable light. Theapyrase present degrades any unreacted dNTP. Light is typically allowedto collect for 3 seconds (although 1-100, e.g., 2-10 seconds is alsosuitable) by a CCD camera linked to the fiber imaging bundle, afterwhich additional wash solution is added to the perfusion chamber toremove excess nucleotides and byproducts. The next nucleotide is thenadded, along with polymerase, thereby repeating the cycle.

During the wash the collected light image is transferred from the CCDcamera to a computer. Light emission is analyzed by the computer andused to determine whether the corresponding dNTP has been incorporatedinto the extended sequence primer. Addition of dNTPs and pyrophosphatesequencing reagents is repeated until the sequence of the insert regioncontaining the suspected polymorphism is obtained.

Example 25 On Picotiter Plate PCR Amplification

Picotiter plate Preparation: In a further embodiment, the singlestranded library attached to beads are distributed directly onto thepicotiter plate and then the nucleic acid template on each bead isamplified (using PCR or other known amplification technology) togenerate sufficient copy number of the template that will generatedetectable signal in the pyrophosphate-based sequencing methodsdisclosed herein.

Example 26 Sequence Analysis of Nucleic Acid on a PTP

Reagents used for sequence analysis and as controls were the fournucleotides and 0.1 μM Pyrophosphate (PPi) were made in substratesolution. Substrate solution refers to a mixture of 300 μM Luciferin and4 μM adenosine 5′-phosphosulfate, APS, which are the substrates for thecascade of reactions involving PPi, Luciferase and Sulfurylase. Thesubstrate was made in assay buffer. The concentration of PPi used totest the enzymes and determine the background levels of reagents passingthrough the chamber was 0.1 μM. The concentration of the nucleotides,dTTP, dGTP, dCTP was 6.5 μM and that of αdATP was 50 μM. Each of thenucleotides was mixed with DNA polymerase, Klenow at a concentration of100 U/mL.

The PTP was placed in the flow chamber of the embodied instrument, andthe flow chamber was attached to the faceplate of the CCD camera. ThePTP was washed by flowing substrate (3 ml per min, 2 min) through thechamber. After this, a sequence of reagents was flown through thechamber by the pump connected to an actuator, which was programmed toswitch positions, which had tubes inserted in the different reagents.The sequence of reagents, flow rates, and flow times were determined.The camera was set up in a fast acquisition mode, with exposure time=2.5s.

The signal output from the pad was determined as the average of countson all the pixels within the pad. The frame number was equivalent to thetime passed during the experiment. Graphing was used to represent theflow of the different reagents.

Example 27 Plate-Based Platform for Picoliterscale PCR Reactions

Materials and Methods

Unless otherwise indicated, all common laboratory chemicals werepurchased either from Sigma (Sigma-Aldrich Corporation, St. Louis,Mich.) or Fisher (Fisher Scientific, Pittsburgh, Pa.).

The PicoTiterPlates™ (25×75×2 mm) were manufactured by anisotropicetching of fiber optic faceplates in a manner similar to that previouslydescribed (Pantano, P. and Walt, D. R., Chemistry of Materials 1996, 8,2832-2835). Plates were etched in three different microwell depths, 26,50 and 76 μm. Microwell center-to-center pitch was 50 μm, and welldiameters ranged between 39 and 44 μm (See FIG. 14), with a calculatedwell density of 480 wells/mm².

Solid-phase immobilization of oligonucleotide primers: Packed beads froma 1 ml NHS-activated Sepharose HP affinity column (Amersham Biosciences,Piscataway, N.J.) were removed from the column and activated accordingto the manufacturer's instructions (Amersham Pharmacia Protocol #71700600AP). Twenty-five microliters of a 1 mM amine-labeled HEG captureprimer (5′-Amine-3 hexaethyleneglycol spacers ccatctgttgcgtgcgtgtc—3′;SEQ ID NO:23) (IDT Technologies, Coralville, Iowa) in 20 mM phosphatebuffer pH 8.0 were bound to the beads. After this, 36 to 25 μm beadswere selected by serial passage through 36 and 25 μm pore filter meshsections (Sefar America, Depew, N.Y.). DNA capture beads that passedthrough the first filter, but were retained by the second were collectedin bead storage buffer (50 mM Tris, 0.02% Tween, 0.02% Sodium Azide, pH8), quantitated with a hemacytometer (Hausser Scientific, Horsham, Pa.)and stored at 4° C. until needed.

Generation of test DNA fragments: Amplification test fragments werederived from a commercially available adenovirus serotype 5 vector,pAdEasy (Stratagene, La Jolla, Calif.). Fragments were amplified usingbipartite PCR primers, the 5′ end of which contained a 20 baseamplification region, and a 20 base 3′ section, complementary to aspecific region of the adenovirus genome. Using these primers, twofragments were amplified from the 12933-13070 and 5659-5767 position ofthe adenovirus genome and assigned labels Fragment A and Fragment B,respectively.

The sequences for the forward and reverse primers for Fragment A was asfollows. A slash (/) denotes the separation between the two regions ofthe primer: forward (5′-cgtttcccctgtgtgccttg/catcttgtccactaggctct-3′;SEQ ID NO:24-SEQ ID NO:25), and reverse(5′-ccatctgttgcgtgcgtgtc/accagcactcgcaccacc-3′; SEQ ID NO:26-SEQ IDNO:27). The primers for the Fragment B included: forward(5′-cgtttcccctgtgtgccttg/tacctctccgcgtaggcg-3′; SEQ ID NO:28-SEQ IDNO:29), and reverse (5′-ccatctgttgcgtgcgtgtc/ccccggacgagacgcag-3′; SEQID NO:30-SEQ ID NO:31).

Reaction conditions included 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1%Triton X-100, 2.5 mM MgCl₂, 0.2 mM dNTP, 1 μM each forward and reverseprimer, 0.1 U/μl Taq (Promega, Madison, Wis.) and 50 nmol template DNA.Both templates were amplified with a PCR program that include 35 cyclesof incubation at 94° C. for 30 seconds, 56° C. for 30 seconds, and 72°C. for 90 seconds. With PCR primers, the total length of the amplifiedfragments was 178 bp for Fragment A and 148 bp for Fragment B.

To generate fluorescent probes, biotinylated double stranded fluorescentprobes were prepared by PCR amplification from the pAdEasy vector asdescribed above. However, the primer sequences were changed to preventhybridization between the test fragment and probe primer regions. Inaddition, the reverse primers for both fragments utilized a 5′biotinfollowed by 3× hexaethyleneglycol spacers to permit productimmobilization to beads prior to elution of the single stranded probe.

The sequence for the forward primer for the fluorescent Fragment A probewas as follows. A slash (/) denotes the separation between the tworegions of the primer(5′-atctctgcctactaaccatgaag/catcttgtccactaggctct-3′; SEQ ID NO:32-SEQ IDNO:33). The sequence for the reverse primer was 5′-biotin—3×hexaethyleneglycolspacers—gtttctctccagcctctcaccga/accagcactcgcaccacc-3′; SEQ ID NO:34-SEQID NO:35. The primers for the Fragment B were as follows: forward(5′-atctctgcctactaaccatgaag/tacctctccgcgtaggcg-3′; SEQ ID NO:36-SEQ IDNO:37), and reverse (5′-biotin-3× hexaethyleneglycolspacers—gtttctctccagcctctcaccga/ccccggacgagacgcag-3′; SEQ ID NO:38-SEQID NO:39).

Fluorescent moieties were incorporated through the nucleotide mixture.This included 0.2 mM dATP/dGTP/dCTP, 0.15 mM TTP and 0.05 mM Alexa Fluor488-dUTP (Molecular Probes, Eugene, Oreg.) for Fragment A. Alternately,0.2 mM dATP/dGTP/TTP, 0.15 mM dCTP and 0.05 mM Alexa Fluor 647—dCTP(Molecular Probes, Eugene, Oreg.) was used for amplifying Fragment B.The fluorescent products were purified with a QIAquick PCR PurificationKit (Qiagen, Valencia, Calif.). The biotinylated DNA was subsequentlybound to 100 μl (approximately 8.1 million) Streptavidin Sepharose HighPerformance beads (Amersham Biosciences) in 1× binding wash (5 mM TrisHCl pH 7.5, 1 M NaCl, 0.5 mM EDTA, 0.05% Tween-20) for 2 hours at roomtemperature. After incubation, the beads were washed three times in TEbuffer (10 mM Tris, 1 mM EDTA, pH 8.0) and incubated with 250 μl meltsolution (0.125 N NaOH/0.1 M NaCl) for 2 minutes, releasing the singlestranded probe from the beads.

Beads were pelleted with brief centrifugation in a benchtop centrifugeand the supernatant was neutralized in 1.25 ml buffer PB (Qiagen) with1.9 μl glacial acetic acid. This mixture was repurified on a QiaQuickcolumn (Qiagen), and the concentration of the purified probe wasdetermined by TaqMan quantification using the BioRad iCycler (BioRad,Hercules, Calif.).

Solution-phase PTPCR was performed as follows. The PCR reaction mixturewas loaded into individual wells of a single 14 mm×43 mmPicoTiterPlate™. For this, 500 μl of PCR reaction mixture (1× PlatinumHiFi Buffer (Invitrogen, Carlsbad, Calif.), 2.5 mM MgSO₄, 0.5% BSA, 1 mMdNTPs (MBI Fermentas, Hanover, Md.), 1 μM forward(5′—cgtttcccctgtgtgccttg—3′; SEQ ID NO:40) and reverse(5′—ccatctgttgcgtgcgtgtc—3′; SEQ ID NO:41) primers, 0.05% Tween-80, 1U/μl Platinum High Fidelity DNA Polymerase (Invitrogen), 0.003 U/μlThermostable Pyrophosphatase (USB, Cleveland, Ohio), and a calculated 5copies of Fragment B template per well) were combined in a 1.5 mlmicrocentrifuge tube. The tube was vortexed thoroughly and stored on iceuntil the PicoTiterPlate™ loading cartridge was assembled.

The in-house PicoTiterPlate™ loading cartridge was attached to thePicoTiterPlate™ with two plastic clips, seating the silicon cartridgegasket firmly on the PicoTiterPlate™ surface (see FIG. 20). The PCRreaction mix was drawn into a 1 ml disposable syringe, and the mouth ofthe syringe inserted into the input tube of the loading cartridge. Theloading cartridge was placed on end, so that the input port was orientedat the bottom of cartridge, and the PCR mix was slowly loaded into thechamber. While loading, inspected through the transparent back of thePicoTiterPlate™ to ensure even, bubble-free delivery.

After loading, the PCR mixture was allowed to incubate for 5 minutes, atwhich time the reaction mixture was withdrawn from the PicoTiterPlate™loading cartridge. The PicoTiterPlate™ was removed from the loadingcartridge, and immediately placed in the amplification chamber (see FIG.21). The PicoTiterPlate™ surface was covered with a 0.25 mm thick SilpadA-2000 silicon sheet (The Bergquist Company, Chanhassen, Minn.). On topof this was placed a 25 mm×75 mm standard glass microscope slide(Fisher). A closed cell foam insulation pad (Wicks Aircraft Supply,Highland, Ill.) was placed on top of the microscope slide. An aluminumlid was attached to the base of the chamber by six 25 mm bolts sealedthe amplification chamber.

Once sealed, the amplification chamber was placed on a Thermocycler MJPTC 225 Tetrad (MJ Research, Waltham, Mass.) equipped with Flat BlockAlpha Units. The amplification program included incubation for 3 minutesat 94° C. (Hotstart Initiation) followed by 40 cycles of incubation for12 seconds at 94° C., 12 seconds at 58° C., 12 seconds at 68° C., with a10° C. final hold. After completion of the PCR program, thePicoTiterPlate™ was removed from the amplification chamber, and theloading cartridge was reattached. A disposable syringe was used to fillthe cartridge chamber with 1 ml of H₂O, and allowed to incubate for 20minutes 10° C. at room temperature.

After the incubation was completed, the recovery solution was withdrawnfrom the loading cartridge and transferred to a 1.5 ml microcentrifugetube. PCR product was quantitated using an iCycler RealTime PCR unit(BioRad) and FAM-labeled reporter probes (Epoch Biosciences, Bothell,Wash.). The TaqMan Universal PCR MasterMix (Applied Biosystems, FosterCity, Calif.) was combined with 0.3 μM forward and reverse primers, 0.15μM FAM-labeled probe, and 27 μl of the reaction mix added to each wellof a 96 well PCR plate.

Purified fragments were used to create a standard curve (six standardsranging from 1×10⁹ to 1×10⁴ molecules per well), which was run intriplicate. The PCR amplification was run with the following parameters:incubation for 5 minutes at 94° C. (hotstart initiation), 60 cycles ofincubation for 15 seconds at 94° C., 45 seconds at 68° C., with a finalhold at 4° C. Data was analyzed using the iCycler Optical SystemsSoftware Version 2.3 (BioRad), and the PCR yield was quantitated usingthe iCycler data and Microsoft Excel (Microsoft, Redmond, Wash.).

Solid-phase PTPCR was performed similarly to solution phase PTPCR,except that DNA capture beads were loaded into the PicoTiterPlate™ wellsprior to amplification by centrifugation as described below. Inaddition, the PCR mixture was loaded into the microwells after the beaddeposition was completed. To facilitate retention of the capture beadsduring wash steps, the solid phase experiments utilized 50 μm deepPicoTiterPlate™s. The PicoTiterPlate™ was placed in an in-house builtplexiglass bead loading jig. This was similar to the PicoTiterPlate™loading jig described in FIG. 20, except that the PicoTiterPlate™ wassandwiched between a bottom Plexiglas plate and a jig top plate,containing inlet and outlet ports, and sealed via a silicon gasket withplastic screws.

Template DNA was preannealed to the DNA capture beads at 5 templatecopies per bead by incubation at 80° C. for 3 minutes, after which beadswere allowed to cool to room temperature for 15 minutes. The beads werethen spun into the PicoTiterPlate™ wells prior to loading the PCRreaction mixture. Bead Loading Buffer (450 μl; 1× Platinum HiFi PCRbuffer (Invitrogen), 0.02% Tween-80) containing one hundred thousandSepharose DNA capture beads (approximately 1 bead per 3 PicoTiterPlate™wells) were injected by pipette into the jig through one of the inletports. Each inlet hole was then sealed with a circular adhesive pad (3MVHS, St. Paul, Minn.). The jig held the PicoTiterPlate™ with its wellsfacing up and covered with the bead suspension. This was centrifuged at2000 rpm for 5 minutes at room temperature in an Allegra 6 centrifuge(Beckman Coulter, Fullerton, Calif.) using a Microtiter Rotor.

After centrifugation, the PicoTiterPlate™ was removed from the jig. ThePCR reaction mix was loaded onto the PicoTiterPlate™ as described forsolution phase PCR. However, the solid-phase PCR mixture omittedtemplate since the template was preannealed to the DNA capture beads.The solid-phase PCR amplification program included additionalhybridization/extension cycles to compensate for the slower kinetics ofthe immobilized primer. The program included incubation for 3 minutes at94° C. for hotstart initiation, 40 cycles of incubation for 12 secondsat 94° C., 12 seconds at 58° C., 12 seconds at 68° C., followed by 10cycles of incubation for 12 seconds at 94° C., 10 minutes at 68° C. forhybridization and extension, with a 10° C. final hold.

Upon completion of the PCR program, the PicoTiterPlate™ was removed fromthe amplification chamber, and washed with 1 ml H₂O as described forsolution phase PCR. The PicoTiterPlate™ was then prepared forhybridization detection of immobilized PCR product.

Hybridization was performed with fluorescently labeled probes asfollows. After PTPCR was complete, the strand complementary to theimmobilized strand was removed. For this, the whole PicoTiterPlate™ wasincubated in 0.125 M NaOH for 8 minutes at room temperature. Thissolution was neutralized by two 5 minute washes in 50 ml of 20 mMTris-acetate pH 7.5. The PicoTiterPlate™ was then placed in acustom-made 800 μl hybridization chamber, and blocked with hybridizationbuffer (3.5×SSC, 3.0% SDS, 20×SSC buffer is 3 M NaCl; 0.3 M Na₃-citrate)at 65° C. for 30 minutes. The contents of the chamber were replaced withfresh hybridization buffer containing the probes: 20 nM fluorescentFragment A (Alexa-488) and Fragment B (Alexa-647). The probes wereallowed to hybridize to their targets. Incubation was carried out at 65°C. for 4 hours while shaking at 200 RPM on an orbital shaker (BamsteadInternational, Dubuque, Iowa).

After hybridization, the PicoTiterPlate™ was washed with 2×SSC, 0.1% SDSfor 15 minutes at 37° C., followed by a 15 minute wash in 1×SSC at 37°C., with two final 15 minute washes in 0.2×SSC at 37° C. Followingpost-hybridization washing, the PicoTiterPlates™ were air dried andplaced in a FLA-8000 Fluorescent Image Analyzer (Fujifilm MedicalSystems USA, Stamford, Conn.) and scanned at the 635 and 473 nmwavelength. The resulting 16-bit tiff images were imported into Genepix4.0 (Axon Instruments, Union City, Calif.). A block of 100 analysisfeatures was drawn over the area of interest and the 635 and 473fluorescence intensities were recorded for each feature. Data was thenexported to Microsoft Excel for further analysis.

Control beads were prepared as follows. Biotinylated test templates Aand B were prepared by PCR amplification from the pAdEasy vector,purified, immobilized on Streptavidin Sepharose High Performance beadsand strand separated as described under “Preparation of FluorescentProbes”. However, fluorescently labeled dNTPs were omitted in the PCRreaction. Pelleted beads were washed 3 times with TE buffer and storedat 4° C. in TE until deposition onto the PicoTiterPlate™.

Results

Solution-phase amplification was demonstrated by loading PicoTiterPlateswith PCR master mix containing a calculated 5 template copies perPicoTiterPlate™ well. Reactions were run in duplicate in PicoTiterPlateswith 26, 50 and 76 μm deep wells. Forty cycles of PTPCR amplificationwere performed as described in Material and Methods. Additives wereincorporated to prevent the deleterious surface effects routinelyreported with silica reaction vessels (Kalinina, O., et al., NucleicAcids Res. 1997, 25, 1999-2004; Wittwer, C. T. and Garling, D. J.,Biotechniques 1991, 10, 76-83; Taylor, T. B., et al., Nucleic Acids Res.1997, 25, 3164-3168).

The inclusion of 0.5% BSA and 0.05% Tween-80 in the reaction mix was notonly effective at reducing surface effects, it also facilitatedamplification. Reducing the relative concentrations of either reagenthad a negative effect on amplification. In addition, due to thepolymerase-inactivating properties of silica surfaces (Taylor, T. B., etal., Nucleic Acids Res. 1997, 25, 3164-3168; Shoffner, M. A., Cheng, J.,Hvichia, G. E., Kricka, L. J. and Wilding, P., Nucleic Acids Res. 1996,24, 375-379), elevated Taq concentrations proved beneficial.Concentrations above 1 U/μl were optimum for enhancing amplicon yield.

Following PTPCR, the solution from each PicoTiterPlate™ was recoveredand triplicate samples of each solution were quantified by TaqMan assay.A standard curve of diluted template (linear from 1×10⁹ to 10⁴molecules, r²=0.995) was used to determine the concentration of theamplified product. The number of molecules amplified per well wasobtained by dividing the amount of amplified product by the total numberof wells in a PicoTiterPlate™ (372,380). The amount of amplification perwell was calculated by dividing this number by the initial templateconcentration per well. PTPCR amplification was successful in all of thePicoTiterPlate™, with yields ranging from 2.36×10⁶ fold in the 39.5 plwells to 1.28×10⁹ fold in the 50 pl wells (See Table below).

Pico Average Fold Fold Final TiterPlate Well Amplification AmplificationProduct Depth [μm] Volume [pl] N = 6 SD Conc. [M] 26 39.5 2.36E+061.02E+06 4.96E−07 50 76.0 1.28E+09 1.03E+09 1.40E−04 76 115.6 9.10E+084.95E+08 6.54E−05

The table shows PicoTiterPlate™ PCR amplification as determined byTaqMan Assay. Values reflect triplicate measurements taken fromduplicate PicoTiterPlates. (N=6); SD=standard deviation.

Yield was influenced by well volume. The concentration of final productobtained for the 50 μm deep wells (1.4×10⁴ M) was significantly greater(p value for ANOVA=0.023) than that obtained in the 76 μm (6.54×10⁻⁵ M)deep wells, both were two orders of magnitude greater than the yieldachieved in the 26 μm deep wells (4.96×10⁻⁷ M). The 50 μm deep microwellyield represented the optimal balancing of the costs and benefitsassociated with low-volume PCR. In this case, maximum elevation of theeffective concentrations and low thermal mass of the reagents wereobtained, but the surface to volume ratio was still low enough toprevent detrimental surface effects from significantly reducingamplification efficiency.

The final concentration of PTPCR product obtained in each of thedifferent well depths (4.96×10⁻⁷ to 1.4×10⁻⁴M) exceeded the 10⁻⁸ Mconcentration typically reported as the maximum achievable before thePCR plateau effect occurs (Sardelli, A., Amplifications 1993, 9, 1-5).The higher effective concentration of primers and template moleculesresulting from the low microwell volume increased the overall reactionefficiency and postponed the onset of the plateau phase until a highermolar yield was achieved. Alternatively, this effect was caused by thehigh concentration of Taq used in the PTPCR reactions, as elevatedpolymerase concentration has also been shown effective in delaying theplateau effect (Kainz, P., Biochim. Biophys. Acta 2000, 1494, 23-27;Collins, F. S., et al., Science 2003, 300, 286-290). The amplificationefficiency over 40 cycles was 44.3, 68.9 and 67.5% for the 26, 50 and 76μm deep wells respectively, providing a high final concentration ofamplicons. The greatest yield was observed in the 50 μm deep wells. Itshould be recognized, however, that cycle number optimization was notconducted; similar amplification yields could likely have been achievedwith far fewer cycles, thereby increasing the efficiency of the PTPCRamplification.

The experimental strategy for clonal solid phase PTPCR, starting with asingle effective copy of a single stranded DNA fragment, and finishingwith a specific bead-immobilized DNA amplicon detected by fluorescentprobe hybridization, is depicted in FIG. 22 and described in detailbelow:

Stage 1: Each PicoTiterPlate™ well contains PCR reaction mix consistingof a single stranded template molecule (either single stranded andannealed to the DNA capture beads, as shown here, or free-floating insolution), Forward “F” (red) and Reverse “R” (blue) primers in solution,as well as R primers attached to a DNA capture bead. Solution phaseprimers are present in an 8:1 molar ratio, with the F primer in excess.Arrows indicate the 5′→3′ DNA orientation.

Stage 2: The initial thermal cycle denatures the DNA template, allowingR primers in solution to bind to the complementary region on thetemplate molecule. Thermostable polymerases initiate elongation at theprimer site (dashed line), and in subsequent cycles, solution-phaseexponential amplification ensues. Bead immobilized primers are notassumed to be major contributors to the amplification at this stage.

Stage 3: Early Phase PCR. During early exponential amplification (1 to10 cycles) both F and R primers amplify the template equally, despite anexcess of F primers in solution.

Stage 4: Mid Phase PCR. Between cycles 10 and 30, the R primers aredepleted, halting exponential amplification. The reaction then enters anasymmetric amplification phase, and the amplicon population becomesincreasingly dominated by F strands.

Stage 5: Late Phase PCR. After 30 to 40 cycles, asymmetric amplificationcontinues to increase the concentration of F strands in solution. ExcessF strands, without R strand complements, begin to anneal tobead-immobilized R primers. Thermostable polymerases utilize the Fstrand as a template to synthesize an immobilized R strand of theamplicon.

Stage 6: Final Phase PCR. Continued thermal cycling forces additionalannealing to bead-bound primers. Solution phase amplification may beminimal at this stage, but concentration of immobilized R strandscontinues to increase.

Stage 7: The non-immobilized, F strand, complementary to the immobilizedR strand, is removed by alkali denaturation. The DNA capture beads arenow populated by single stranded R strands of the amplicon.

Stage 8: Fluorescently labeled probes (green bars) complementary to theR strand are annealed to the immobilized strand. Probes specific forparticular strand sequences are labeled with unique fluorophores,resulting in a range of homogenous and heterogeneous fluorescent signalsdepending on the number of discrete templates amplified within a givenPicoTiterPlate T well.

Initially, fluorescently labeled probe specificity was confirmed bybinding biotinylated Fragment A or Fragment B test DNA fragments tostreptavidin Sepharose beads, loading the beads into a 50 μm deepPicoTiterPlate™ by centrifugation and hybridizing a mixed population offluorescently labeled probes for the Fragment A and Fragment Bfragments. No mixed signals or nonspecific hybridizations were observed;the beads with the Fragment A product displayed the 488 nm signal, whilethe Fragment B beads exhibited the 635 nm signal (See FIGS. 23A and23B). Close examination of FIGS. 23A and 23B reveals a few Fragment Abeads in the Fragment B pad and vice versa. Given the purity of thesignal displayed by these nomadic beads, it is likely that they areeither the product of some cross contamination during the loadingprocess, or were washed from one pad to the other during subsequent washsteps.

As indicated in FIG. 23C, the fluorescent probes detected successfulsolid phase PTPCR amplification of both Fragment A and Fragment Btemplates. The signals generated by the hybridized probe depended on therelative efficiency of dye incorporation within the probes, thesensitivity of the reactions to unequal amounts of template DNA, as wellas the total and relative amounts of amplified product present on eachbead. In addition, it is likely that the amount of template generatedand retained on the DNA capture beads varied from well to well, and thenumber of capture primers bound to each bead is also likely to vary dueto bead size distribution. As a result, the non-normalized ratiosgenerated by the probe hybridization should be seen as semi-qualitativerather than quantitative data. Nevertheless, the fluorescent signalsgenerated by the hybridized probes ranged from a homogeneous Fragment Bsignal (red) to an equally homogenous Fragment A signal (green), withheterogeneous mixes of the two signals (degrees of yellow) evident aswell.

Due to the probe specificity displayed by the controls, as well as thesizeable number of homogenous red and green beads on thePicoTiterPlate™, it is unlikely that nonspecific probe hybridizationcaused the heterogeneous signals. The close proximity of homogenousbeads of either template suggests it is unlikely that the heterogeneousbeads resulted from amplicon leakage between wells during amplification;if intra-well cross-talk were responsible, one would expect to seeheterogeneous beads located between homogenous beads of either template,and a generally patchy distribution of homogenous signals. Rather, it islikely that template molecules disassociated from their original beadand reannealed to new beads in the PicoTiterPlate™ loading mix prior tobeing spun into the microwells, or were washed from one bead to anotheras the PCR mix was applied to the PicoTiterPlate™. Regardless of thecause of the mixed template beads, the hybridization results show thatPCR amplification in the PicoTiterPlate™ microwells can drive sufficientproduct to the DNA capture beads to enable fluorescent probehybridization and detection.

Discussion

The results in this example demonstrate that PicoTiterPlate™-based PCRalleviates many factors associated with the DNA amplification process,such as high costs of reagents, large numbers of reactions, and lengthyreaction times, delivering another “evolutionary jump” in PCRtechnology. The microwells on a single PicoTiterPlate™ can function asup to 370,000 discrete reaction vessels achieving high yield (2.3×10⁶ to1.2×10⁹ fold) amplification even at reaction volumes as low as 39.5picoliters. As a result, throughput is increased, and the total reagentcost for PTPCR is reduced; the reaction volume contained in an entire 26or 76 μm deep PicoTiterPlate™ is 15.3 and 43 μl, respectively. Increasesin the size of the PicoTiterPlate™ can further increase the maximalthroughput. For example, increasing the PicoTiterPlate™ dimensions to 40mm×75 mm provides approximately 1.4×10⁶ discrete reaction vessels, and aPicoTiterPlate™ possessing the same perimeter dimensions as acommercially available 96-well PCR plate (85.47 mm×127.81 mm) couldcontain as many as 5.24×10⁶ wells.

Solution phase PCR amplifications, regardless of the number and volumein which they are conducted, are of limited utility unless the productcan be recovered easily and efficiently. Previous efforts in parallelPCR (Nagai, H., et al., Anal. Chem. 2001, 73, 1043-1047) requiredevaporation of the liquid reaction mixture, leaving the amplicon driedto the walls of the microreactor, after which it could be recovered forfurther manipulations. The methodology disclosed herein avoids theproblems of product recovery by including solid phase amplification,immobilizing the PCR product to a DNA capture bead. Thus, the product ofa PicoTiterPlate™ microwell reaction is not 370,000 wells containingsolution-phase PCR product, but up to 370,000 beads bound withimmobilized PCR product. These PCR products are suitable for numeroussolid-phase methods of nucleic acid interrogation including thepotential capacity to support a massively parallel approach tosequencing whole genomes containing up to hundreds of millions of bases.The simplicity of the disclosed method would drastically reduce costsfor sequencing and other applications now requiring robotics to maintainlarge-scale cloning and PCR.

The disclosures of one or more embodiments of the invention are setforth in the accompanying description. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims. In the specification and the appended claims, the singular formsinclude plural referents unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless expressly statedotherwise, the techniques employed or contemplated herein are standardmethodologies well known to one of ordinary skill in the art. Theexamples of embodiments are for illustration purposes only. All patentsand publications cited in this specification are incorporated byreference.

Example 28 Rig Sequencing Method

Step 1: Preparation of pAdEasy PCR DNA Beads

This procedure was used for a 384-well plate PCR of Adenovirus clones.Streptavidin-Sepharose beads (12 mls) were prepared for binding PCRfragments by washing once with 2 M NaCl solution and resuspending in 288mls of 2 M NaCl. The washed beads were transferred to fifteen 96-wellplates at 200 μl of bead suspension/well. The PCR products (25 μl) weretransferred to a 384-deep-well plate using a Tecan TeMo robot. To bindDNA to solid supports, 25 μl of bead suspension (15,000 beads) wereadded to each well of every 384-deep well plate using a Tecan TeMo robotand mixed. The final concentration of NaCl in the binding reaction was 1M. The binding reaction was incubated with shaking at room temperaturefor 3 hr on a shaker. The contents of the microtiter plates were pooledby inverting the 384-well plates on to a reservoir and centrifuging at1000 g in a Beckman Allegra benchtop centrifuge. The pooled beads weretransferred into a 50 ml Falcon tube, centrifuged at 1000 g, and thesupernatant was removed.

Approximately a million beads (mobile solid support) were washed oncewith 100 μl of 2 M NaCl followed by two washes with distilled water (100μl each). The washed beads were incubated in 300 μl melting reagent (0.1M NaCl and 0.125 M NaOH) for 10 minutes in a rotator to remove thenon-biotinylated DNA strand. The tube was centrifuged at maximum speedto pellet the beads and the melt solution was removed and discarded. Thebeads were washed with 100 μl of melt solution followed by three morewashes with 1× Annealing buffer. After the washes, the beads wereresuspended in 25 μl of 1× Annealing buffer.

Primer P2 (500 pmoles) was added to the bead mixture and mixed. The beadmixture, in tubes, was placed into an automated incubator (PCRthermocycler in this case) with the following temperature profiles:incubation at 60° C. for 5 minutes, decrease 0.1° C./second down to 50°C., incubation at 50° C. for 5 minutes, decrease 0.1° C./second down to40° C., incubation at 40° C. for 5 minutes, decrease 0.1° C./second downto 4° C., incubation at 4° C. forever.

After annealing, the beads were washed carefully and resuspended in 200μl of Bst DNA polymerase binding solution. Then, 10 μl aliquots (50,000beads) of the bead suspension were processed for sequencing on theinstrument described below.

Step 2: Preparation of Control DNA Beads

Six control DNA sequences TF 2, 7, 9, 10, 12 and 15 were cloned intopBluescript II KS+vector and plasmid DNA was used as template for PCRwith one biotinylated primer for solid-phase immobilization of theamplicons.

The following reagents were added to a 1.7 ml tube to create a PCR mix.

10 X HIFI buffer 100 μl  10 mM dNTP mix 100 μl  50 mM MgSO₄ 60 μl5′-Bio-3HEG-MMP1B 10 μl MMP1A 10 μl HIFI Taq Polymerase 10 μl Mol. Bio.Grade Water 690 μl 

Twenty microliters of plasmid template DNA was added and the mix wasaliquoted by 50 μl into 0.2 ml PCR tubes. The following program was usedfor thermocycling: incubation at 94° C. for 4 minutes; 39 cycles ofincubation at 94° C. for 15 seconds, 58° C. for 30 seconds, 68° C. for90 seconds, and 68° C. for 120 seconds; hold at 10° C.

Amplified DNA for each test fragment was purified using the QiagenMinElute PCR Clean-Up Kit as per manufacturer's instructions. The purityand yield of each of the test fragments DNA was assessed using theAgilent 2100 Bioanalyzer and DNA 500 reagent kit and chip. BiotinylatedPCR products were immobilized onto Sepharose streptavidin beads at 10million DNA copies/bead.

Beads were washed once with 2 M NaCl solution. This was done by adding100 μl, vortexing briefly to resuspend the beads, centrifuging for 1minute at maximum speed to pellet the beads, and then removing thesupernatant. This was followed by a second wash with 2 M NaCl. The beadswere then resuspended in 30 μl of 2 M NaCl. PCR product was added tobeads. The mixture was vortexed to resuspend the beads in solution andthen placed in a rack, on a titer plate shaker, at speed 7, for 1 hourat room temperature.

The non-biotinylated second strand was removed by incubation with thealkaline melt solution (0.1 M NaOH/0.15 M NaCl) for 10 minutes in anoverhead rotator at room temperature. This was followed by washing thebeads once with 100 μl of melt solution and three times with 100 μl of1× annealing buffer (50 mM Tris-Acetate, pH 7.5; 5 mM MgCl₂). Sequencingprimer was annealed to the immobilized single-stranded DNA bycentrifugation for one minute at maximum speed. The supernatant wasremoved and the beads were resuspended in 25 μl of 1× annealing buffer.Next, 5 μl of sequencing primer MMP7A (100 μmol/μl) was added to thebead suspension and the following temperature profile was used tohybridize the sequencing primer:

-   -   Incubation at 60° C. for 5 minutes;    -   Decrease 0.1° C./second down to 50° C.;    -   Incubation 50° C. for 5 minutes;    -   Decrease 0.1° C./second down to 40° C.;    -   Incubation at 40° C. for 5 minutes;    -   Decrease 0.1° C./second down to 4° C.; and    -   Hold at 4° C.

Beads were washed twice with 100 μl of 1× annealing buffer and thenresuspended to a final volume of 200 μl with 1× annealing buffer andstored in 10 μl aliquots in labeled tube strips in a 4° C. refrigerator.

Step 3: Sequencing Chemistry

Sepharose beads with immobilized single stranded DNA templates andannealed sequencing primer were incubated with E. coli single strandbinding protein (Amersham Biosciences) (5 μl of 2.5 μg/μl ssb stocksolution per 50,000 beads) and 500 U (10 μl of 50 U/μl) of Bst DNApolymerase (NEB) in 200 μl of Bst polymerase binding solution (25 mMTricine pH 7.8; 5 mM magnesium acetate; 1 mM DTT; 0.4 mg/ml PVP MW360,000) for 30 minutes at room temperature on a rotator. After this,the DNA beads were mixed with the SL beads and deposited into the wellsof the PicoTiter Plate as follows. Reagents required for a sequencingrun on a 454 instrument included 1) substrate wash solution; 2) apyrasecontaining wash solution; 3) 100 nM inorganic pyrophosphate calibrationstandard; 4) individual nucleotide triphosphate solutions.

All solutions were prepared in the sulfurylase-luciferase assay bufferwith enzyme substrates (25 mM Tricine pH 7.8; 5 mM magnesium acetate;0.4 mg/ml PVP MW 360,000; 0.01% Tween 20; 300 μM D-luciferin; 4 μM APS).The substrate wash solution was identical to the luciferase assaybuffer. The apyrase containing wash solution was based on the luciferaseassay buffer, except no enzyme substrates (APS and D-luciferin) wereadded and this wash contained apyrase (Sigma St. Lous, Mo.;Pyrosequencing AB, Pyrosequencing, Inc. Westborough, Mass.) in the finalconcentration of 8.5 U/1.

Sodium pyrophosphate (PP_(i)) standard was prepared by adding sodiumpyrophosphate tetrabasic decahydrate (Sigma, St. Louis, Mo.) to theluciferase assay buffer to a final concentration 100 nM. Nucleotidetriphosphates (dCTP, dGTP, TTP; minimum diphosphate grade) (AmershamBiosciences AB, Uppsala, Sweden) were diluted to final concentration of6.5 μM in the luciferase assay buffer. Deoxyadenosine triphosphateanalog, 2′-Deoxyadenosine-5′-O-(1-thiotriphosphate), Sp-isomer(Sp-dATP-α-S, Biolog Life Science Institute, Bremen, Germany) wasdiluted to final concentration of 50 μM in the luciferase assay buffer.

Step 4: Cloning His6-BCCP-sulfurylase and His6-BCCP-luciferase

Bacillus stearothermophilus (Bst) ATP sulfurylase (E.C. 2.7.7.4) andfirefly (Photinus pyralis) luciferase (E.C. 1.13.12.7) were cloned intoNhe I-BamH I digested pRSET-A vector (Invitrogen). The coding sequenceof the BCCP (biotin carboxyl carrier protein) gene (Alix, J. H., DNA 8(10), 779-789 (1989); Muramatsu, S, and Mizuno, T., Nucleic Acids Res.17 (10), 3982 (1989), Jackowski, S, and Alix, J. H., J. Bacteriol. 172(7), 3842-3848 (1990); Li, S. J. and Cronan, J. E. Jr., J. Biol. Chem.267 (2), 855-863 (1992), Genbank accession number M80458) was used todesign PCR primers to amplify the fragment corresponding to amino acids87-165 of the BCCP protein. The forward primer was5′-ctagctagcatggaagcgccagcagca-3′; SEQ ID NO:42 and the reverse primerwas 5′-ccgggatccctcgatgacgaccagcggc-3′; SEQ ID NO:43. The PCR cocktailwas prepared as Mix 1 and Mix 2, 25 μl each. Mix 1 included 75 pmoles ofprimers, 100 ng of E. coli genomic DNA and 5 μmoles of dNTPs. Mix 2included 1 unit of Fidelity Expand DNA polymerase (BoehringerMannheim/Roche Diagnostics Corporation, Indianapolis, Ind., Cat. No. 1732 641) and 5 μl of 10× Fidelity Expand buffer (BoehringerMannheim/Roche Diagnostics Corporation, Indianapolis, Ind.). To allowPCR hot-start, Mix 1 and Mix 2 were heated separately for 20 seconds at96° C. before they were pooled. The pooled reaction was cycled asfollows: incubation at 96° C. for 3 min, 10 cycles of incubation at 96°C. for 30 sec, 55° C. for 1 min, and 68° C. for 2 min, then 20 cycles ofincubation at 96° C. for 30 sec, 60° C. for 1 min, and 68° C. for 2 min,followed by a polishing step of incubation at 72° C. for 7 min. AfterPCR, a single 250 bp fragment was obtained. The BCCP fragment wasdigested with Nhe I and BamH I and subcloned into Nhe I-BamH I digestedpRSET-A.

Step 5: Expression of Sulfurylase and Luciferase.

The Bst ATP sulfurylase and P. pyralis luciferase open reading frameswere amplified by PCR with primers that contain Pst I/Hind III and BamHI/Xho I sites (the first enzyme was at the 5′ end and the second enzymewas at the 3′ end), respectively. This produced an N-terminal fusion of6× His and BCCP domain to ATP sulfurylase and luciferase. The enzymeswere expressed in E. coli using biotin-supplemented growth media toallow for in-vivo biotinylation via the BCCP domain. The enzymes werepurified to near homogeneity using a combination of IMAC and asize-exclusion column chromatography. Purification was assessed byelectrophoresis using the Agilent 2100 Bioanalyzer on Protein 200 Pluschips.

Step 6: Solid Phase Immobilization of Luciferase and Sulfurylase

The enzymes were immobilized onto Dynal M-280 streptavidin coatedmagnetic microparticles (Dynal, Oslo, Norway) and Bangs microspheres(300 nm) by incubation of 1:3 mixture of ATP sulfurylase and luciferase,respectively. The binding was performed by mixing 50 μg of ATPsulfurylase and 150 μg of luciferase with 1 mg of Dynal M-280 beads or0.6 mg of Bangs microspheres in TAGE buffer (25 mM Tris-Acetate pH 7.8,200 mM ammonium sulfate, 15% v/v glycerol and 30% v/v ethylene glycol).The mixture was incubated for 1 hour at 4° C. on a rotator. Afterbinding, the beads could be stored at −20° C. in the enzyme solution for3 months. Before use, beads were washed thoroughly in luciferase assaybuffer containing 0.1 mg/ml bovine serum albumin (Sigma, St Louis, Mo.).Immobilized enzyme activity was assayed using a luminometer (Turner,Sunnyvale, Calif.). Washed beads were stored on ice until depositiononto a PTP slide.

Step 7: PicoTiterPlates™ (PTPs)

The PicoTiterPlates™ (25×75×2 mm) were manufactured by anisotropicetching of fiber optic face plates in a manner similar to that describedin literature. Plates were etched in three different microwell depths,26, 50 and 76 mm. Microwell center-to-center pitch was 50 μm, and welldiameters ranged between 39 and 44 μm with a calculated well density of480 wells/mm².

Step 8: PTP Loading

Sepharose beads carrying DNA templates and Dynal M-280/Bangs 0.3 μm beadmixture with immobilized sulfurylase and luciferase enzymes weredeposited into individual wells of a PicoTiter plate using acentrifugation-based method. The procedure employed an in-housepolycarbonate fixture (jig) which included a bottom plate (with slidepositioning pegs), an elastomer sealing gasket, and a top plate with twoloading ports. The PTP slide was placed onto the bottom plate with theetched side facing up and the top plate with sealing gasket in place wasclamped on top of the PTP slide. The whole assembly was tightened withfour plastic screws in order to provide a water-tight seal. The sealinggasket was designed to form a mask for bead deposition, resulting in onehexagonal area (14×43 mm) covering roughly 270,000 PTP wells.

Beads were deposited in ordered layers. The PTP was removed fromincubating in Bead Wash Buffer. Layer 1, a mix of DNA and enzyme beads,was deposited. After centrifuging, Layer 1 supernatant was aspirated offthe PTP and Layer 2, Dynal enzyme beads, was deposited.

A bead suspension was prepared by mixing 150,000 DNA carrying Sepharosebeads in 120 μl of the ssb/Bst pol binding mix (see above) with 270 μlof Dynal-SL and Bangs-SL beads (both at 10 mg/ml) in a total volume of500 μl of the luciferase assay buffer containing 0.1 mg/ml bovine serumalbumin. The bead slurry was vortexed and flowed into the beaddeposition jig through pipetting ports. Care was taken to avidintroducing air bubbles. The jig/PTP assembly was centrifuged at 2000rpm for 8 minutes in a Beckman Allegra 6 centrifuge equipped with a4-position plate swing-out rotor. After centrifugation, the supernatantwas carefully removed from the jig chamber using a pipette. A secondlayer of only Dynal-SL beads was deposited. This layer included 125 μlof Dynal-SL (at 10 mg/ml) and 375 μl Bead Wash Buffer in a 1.5 ml tube(2.5 mg/ml Dynal beads). The Dynal bead mixture was pipetted into thePTP main active area and centrifuged for 8 minutes at 2000 rpm. Layer 2mixture was aspirated and the PTP was placed back into Bead Wash Buffer(luciferase assay buffer with 0.1 mg/ml bovine serum albumin and 8.5 U/lapyrase) until ready to load onto the Sequencer.

Step 9: Sequencing Instrument

The in-house sequencing instrument included three major assemblies: afluidics subsystem, a PTP cartridge/flow chamber, and an imagingsubsystem. The fluidics subsystem included reagent reservoirs, reagentsinlet lines, a multi-valve manifold, and a peristaltic pump. It allowedfor reagent delivery into the flow chamber, one reagent at a time, at apre-programmed flow rate and duration. The PTP cartridge/flow chamberwas designed in such a way that after attaching a PTP, there would be300 μm space between the PTP top (etched side) and the chamber ceiling.It included means for temperature control of the reagents and PTP, aswell as a light-tight housing. The polished side of a PTP was exposed atthe back side of the PTP cartridge and was placed directly in contactwith the imaging system. The imaging system comprised a CCD camera witha 1-1 imaging fiber bundle, as well as cryogenic cooling system for thecamera, and camera control electronics. The camera used was a SpectralInstruments (Tucson, Ariz.) series 600 camera with a Fairchild ImagingLM485 CCD (16 million pixels, 15 μm pixel size). This was bondeddirectly to the imaging fiber bundle with 6 μm fiber pitch. The camerawas cooled to −70° C. and operated in a frame transfer mode. In thisway, the center portion of the CCD was used for imaging while the outerportion of the CCD was used for image storage and read-out. The read-outoccurred through 4 ports at each corner of the CCD. The data acquisitionrate was set to 1 frame per 30 seconds. The frame-transfer shift timewas approximately 0.25 seconds. All camera images were stored in a UTIFF16 format on a computer hard drive (IBM eServer xSeries 335, IBM, WhitePlains, N.Y.).

Step 10: Sequencing Run Conditions

The cyclical delivery of sequencing reagents into the PTP wells andwashing of the sequencing reaction byproducts from the wells wasachieved by a pre-programmed operation of the fluidics system. Theprogram was written in a form of a Microsoft Excel script, specifyingthe reagent name (Wash, dATPαS, dCTP, dGTP, dTTP, PP_(i) standard), flowrate and duration of each script step. Flow rate was set at 3 ml/min forall reagents and the linear velocity within the flow chamber wasapproximately. An initial wash step (5 minutes) was followed by a PP_(i)standard flow (2 min), followed by 21 or 42 cycles of(Wash—C—Wash—A—Wash—G—Wash—T), where each nucleotide flow was 0.5 minuteand wash steps were 2 minutes. After all cycles of nucleotide additionsand washes, a second PP_(i) standard flow (2 min) was delivered,followed by a final 5 minutes wash step. The total run time was 4 hours.Reagent volumes required to complete this run script were as follows:300 ml each wash solution, 50 ml of each nucleotide solution, 20 ml ofPP_(i) standard solution. During the run, all reagents were kept at roomtemperature. Because the flow chamber and flow chamber inlet tubing weremaintained at 3° C., all reagents entering the flow chamber were at 30°C.

References

Hamilton, S. C., J. W. Farchaus and M. C. Davis. 2001. DNA polymerasesas engines for biotechnology. BioTechniques 31:370.

QiaQuick Spin Handbook (QIAGEN, 2001): hypertext transferprotocol://world wideweb.qiagen.com/literature/handbooks/qqspin/1016893HBQQSpin_PCR_mc_prot.pdf.

Quick Ligation Kit (NEB): hypertext transfer protocol://world wideweb.neb.com/neb/products/mod_enzymes/M2200.html.

MinElute kit (QIAGEN): hypertext transfer protocol://world wideweb.qiagen.com/literature/handbooks/minelute/1016839_HBMinElute_Prot_Gel pdf.

Biomagnetic Techniques in Molecular Biology, Technical Handbook, 3rdedition (Dynal, 1998): hypertext transfer protocol://world wideweb.dynal.no/kunder/dynal/DynalPub36.nsf/cb927fbab127a0ad4125683b004b011c/4908f5b1a665858a41256adf005779f2/$FILE/Dynabeads M-280 Streptavidin.pdf.

Bio Analyzer User Manual (Agilent): hypertext transfer protocol://worldwide web.chem.agilent.com/temp/rad31B29/00033620.pdf

BioAnalyzer DNA and RNA LabChip Usage (Agilent): hypertext transferprotocol://world wide web.agilent.com/chem/labonachip

BioAnalyzer RNA 6000 Ladder (Ambion): hypertext transfer protocol:/worldwide web.ambion.com/techlib/spec/sp_(—)7152.pdf

We claim:
 1. A method for sequencing nucleic acids comprising: (a) delivering the nucleic acids to an array of at least 10,000 reaction chambers on a planar -surface, such that a plurality of the reaction chambers comprise beads capable of binding the nucleic acid and no more than one nucleic acid; (b) amplifying the nucleic acids in the reaction chambers, wherein a plurality of amplified copies of the nucleic acids are immobilized to beads; and (c) performing a sequencing reaction simultaneously in a plurality of the reaction chambers comprising the immobilized amplified copies of the nucleic acids.
 2. The method of claim 1 wherein the reaction chambers have a center to center spacing of between 20 to 100 μm.
 3. The method of claim 1 wherein the nucleic acids are between 30-500 bases.
 4. The method of claim 1 wherein the amplifying step is accomplished using polymerase chain reaction.
 5. The method of claim 1 wherein the sequencing reaction is a pyrophosphate-based sequencing reaction.
 6. The method of claim 1 wherein the sequencing reaction comprises the steps of: (a) annealing an effective amount of a sequencing primer to immobilized amplified copies of the nucleic acids and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′ end of said sequencing primer, a sequencing reaction byproduct; and (b) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid in a plurality of the reaction chambers.
 7. The method of claim 1 wherein the reaction chambers are cavities formed by etching one end of a fiber optic bundle. 